Assaying the efficacy of dual-antiplatelet therapy: use of a controlled-shear-rate microfluidic device with a well-defined collagen surface to track dynamic platelet adhesion.

We report the development and demonstration of an assay that distinguishes the pharmacological effects of two widely used antiplatelet therapies, aspirin (COX-1 inhibitor) and clopidogrel (P2Y12 inhibitor). Whole blood is perfused through a low-volume microfluidic device in contact with a well-characterized (ellipsometry, atomic force microscopy) acid-soluble type I collagen surface. Whole human blood treated in vitro with a P2Y12 inhibitor 2-methylthioadenosine 5'-monophosphate triethylammonium salt (2-MeSAMP) extended the time to the start of platelet recruitment, i.e., platelet binding to the collagen surface. Treatment with 2-MeSAMP also slowed the rate of aggregate buildup, with an overall reduced average platelet aggregate area after 8 min of constant blood flow. A far smaller effect was observed for in vitro treatment with aspirin, for which the rate of change of surface coverage is indistinguishable from controls. In whole blood obtained from patients under treatment with dual-antiplatelet therapy (aspirin and clopidogrel), a significant extension of time to platelet recruitment was observed along with a slowed rate of aggregate buildup and an average aggregate size approximately half that of control measurements. Differentiation of the pharmacological effects of these two well-targeted antiplatelet pathways suggests a role for this assay in determining the antiplatelet effects of these and related new therapeutics in clinical settings.

Interactions between multiple cell types in parallel microfluidic channels: monitoring platelet adhesion to an endothelium in the presence of an anti-adhesion drug.

A simple method for immobilizing endothelial cells in the channels of a microfluidic device fabricated with soft lithography is presented that requires no surface oxidation of the substrate material used in conjunction with the microfluidic device and is operable even with a reversible seal. Specifically, optimal conditions for culturing bovine pulmonary artery endothelial cells (bPAECs) to the surface of a Petri dish were investigated. The parameters investigated included fibronectin concentration, temperature, seeding density, and immobilization time. To enhance the utility of the device, all optimization studies, and studies involving platelet adhesion to the immobilized endothelium, were performed in parallel channels, thereby enabling improved throughput over a single channel device. The optimal conditions for cell immobilization included coating the Petri dish with 100 microg/mL fibronectin, a seeding cell density of 1.00 x 10(5) cells mL(-1), and an immobilization time of 90 min at 37 degrees C. The device was then employed to monitor the physical interaction (adhesion) of platelets to the immobilized endothelium in the presence of a known platelet activator (ADP) and a drug inhibitor of platelet activation. The number of platelets adhering to the endothelial cells in the channels increased from 17.0 +/- 2.3 in the absence of ADP to 63.2 +/- 2.4 in the presence of 5.00 microM ADP. Moreover, the data presented here also shows that inhibition of endothelium nitric oxide (NO) production, a recognized inhibitor of platelet adhesion to the endothelium, increased the number of platelets adhering to the surface to 35.4 +/- 1.0. In the presence of NO inhibition and 5.00 microM ADP, the affect on platelet adhesion was further increased to 127 +/- 5.2. Finally, this device was employed to investigate the effect of a drug known to inhibit platelet adhesion (clopidogrel) and, in the presence of the drug, the platelet adhesion due to activation by 5.00 microM ADP decreased to 24.0 +/- 3.8. This work is the first representation of multiple cell types physically interacting in the channels of a microfluidic device and further demonstrates the potential of these devices in the drug discovery process and drug efficacy studies.

Real-time monitoring of adhesion and aggregation of platelets using thickness shear mode (TSM) sensor.

Hemostasis is required to maintain vascular system integrity, but thrombosis, formation of a clot in a blood vessel, is one of the largest causes of morbidity and mortality in the industrialized world. Novel clinical and research tools for characterizing the hemostatic system are of continued interest, and the object of this research is to test the hypothesis that clinically relevant platelet function can be monitored using an electromechanical sensor. A piezoelectric thickness shear mode (TSM) biosensor coated with collagen-I fibers to promote platelet activation and adhesion was developed and tested for sensitivity to detect these primary events. Magnitude and frequency response of the sensor were monitored under static conditions at 37 degrees C, using platelet-rich plasma (PRP), and PRP with adenosine diphosphate (ADP), a clinical aggregation inhibitor (abciximab), or a collagen binding inhibitor. Sensors loaded with PRP exhibited a 3-stage response; no significant change in response for the first 20 min (Stage-1), followed by a larger drop in response (Stage-2) and subsequently, response gradually increased (Stage-3). Exogenous ADP stimulated an immediate Stage-2 response, while abciximab delayed and reduced the magnitude change of Stage-2. In the presence of collagen inhibitor, Stage-2 response was similar to that of control but was delayed by an additional 20 min. The obtained results, supported by epifluorescence and complementary SEM studies, demonstrated the selective sensitivity of TSM electromechanical biosensors to monitor platelet function and inhibition, particularly aggregation.

The intervention of antioxidant therapy on platelet adhesion and immunomodulation in experimental physical stress.

During effort overstress the reactive oxygen species act chiefly on unsaturated lipids, inducing the formation of certain peroxidation products. We have investigated malondialdehide (MDA), platelet adhesion index, and immunological activation parameters during effort overstress and administration of vitamins E and C. Biochemical measurements were performed on erythrocytes and heart homogenate. In the vitamin E supplemented group, the platelet adhesion index was constantly correlated with the MDA level (p < 0.001). There is a protecting effect concerning the oxidative stress in animals pretreated with vitamin E and C, which is expressed through the diminution of the MDA quantity both in the erythrocyte and in the heart. The physical effort required by swimming led to a decrease in the NBT test values and in the activity of the serum complement. The steady administration of vitamin E in the effort overstress, due to its antioxidant properties, causes the progressive decrease in peroxidation and platelet adhesion.

Reduced platelet adhesion to titanium metal coated with apatite, albumin-apatite composite or laminin-apatite composite.

Titanium metal coated with apatite (HA-Ti), albumin-apatite composite (AA-Ti) or laminin-apatite composite (LA-Ti) was prepared by the immersion of NaOH- and heat-treated titanium metal in a calcium phosphate solution, or one supplemented with albumin or laminin. Platelet adhesion to the obtained materials under flow conditions was investigated in real time using a cone- and plate-type viscometer and fluorescence labeled platelets. Adhesion and activation of the platelets on the HA-Ti, AA-Ti and LA-Ti were definitely suppressed as compared with those on untreated titanium metal with a mirror surface. Furthermore, the numbers of platelets adhered to AA-Ti and LA-Ti are smaller than those adhered to HA-Ti, although the differences were not statistically significant. These findings suggest that HA-Ti, AA-Ti and LA-Ti, especially AA-Ti and LA-Ti, would exhibit thromboresistance that is superior to commercially pure titanium metal in terms of platelet adhesion.

Blood compatibility of titanium oxides with various crystal structure and element doping.

BACKGROUND: Titanium oxides are known to be good hemocompatible, therefore they are suggested as coatings for blood contacting implants. But little is known about the influence of physical characteristics like crystal structure, roughness and electronic state on the activation of blood platelets and the blood clotting cascade. METHODS: Titanium oxide films were produced by metal plasma deposition and implantation in the form of rutile, crystalline and nanocrystalline anatase + brookite and amorphous TiO2. The redox potential was reduced by implantation of chromium ions, the Fermi level of the semiconductive oxide was shifted by ion implantation of the electron donor phosphorous. Hemocompatibility was determined by measuring the adhesion of blood platelets, their P-selectine expression, and of the blood clotting time on these samples. RESULTS: The crystalline titanium oxides had a slightly higher activation of the clotting cascade but lower platelet adhesion than nanocrystalline and amorphous titanium oxides. The surface roughness below 50 nm had no obvious effect. Both, implantation of phosphorous or chromium ions, strongly reduced the activation of the clotting cascade, but only the phosphorous implanted surface also showed a reduced platelet activation, whereas platelet adhesion and activation was strongly increased on the chromium implanted surfaces. CONCLUSION: Phosphorous doping of rutile TiO2 can increase its hemocompatibility, both concerning blood platelets and blood clotting cascade, but the biochemical mechanism has to be worked out.

Complementary roles of glycoprotein VI and alpha2beta1 integrin in collagen-induced thrombus formation in flowing whole blood ex vivo.

Platelets interact vigorously with subendothelial collagens that are exposed by injury or pathological damage of a vessel wall. The collagen-bound platelets trap other platelets to form aggregates, and they expose phosphatidylserine (PS) required for coagulation. Both processes are implicated in the formation of vaso-occlusive thrombi. We previously demonstrated that the immunoglobulin receptor glycoprotein VI (GPVI), but not integrin alpha2beta1, is essential in priming platelet-collagen interaction and subsequent aggregation. Here, we report that these receptors have yet a complementary function in ex vivo thrombus formation during perfusion of whole blood over collagen. With mice deficient in GPVI or blocking antibodies, we found that GPVI was indispensable for collagen-dependent Ca2+ mobilization, exposure of PS, and aggregation of platelets. Deficiency of integrin beta1 reduces the GPVI-evoked responses but still allows the formation of loose platelet aggregates. By using mice deficient in G(alpha)q or specific thromboxane A2 and ADP antagonists, we show that these autocrine agents mediated aggregation but not collagen-induced Ca2+ mobilization or PS exposure. Collectively, these data indicate that integrin alpha2beta1 facilitates the central function of GPVI in the platelet activation processes that lead to thrombus formation, whereas the autocrine thromboxane A2 and ADP serve mainly to trigger aggregate formation.

Recombinant factor VIIa enhances deposition of platelets with congenital or acquired alpha IIb beta 3 deficiency to endothelial cell matrix and collagen under conditions of flow via tissue factor-independent thrombin generation.

A novel approach to treat bleeding episodes in patients with Glanzmann thrombasthenia (GT) and perhaps also in patients receiving alpha IIb beta 3 inhibitors is the administration of recombinant factor VIIa (rFVIIa). The mechanism of action of rFVIIa in these patients is, however, still unclear. We studied the effect of rFVIIa-mediated thrombin formation on adhesion of alpha IIb beta 3-deficient platelets under flow conditions. Adhesion of alpha IIb beta 3-deficient platelets to the extracellular matrix (ECM) of stimulated human umbilical vein endothelial cells or to collagen type III was studied using a model system with washed platelets and red cells. When alpha IIb beta 3-deficient platelets were perfused over the surface at arterial shear rate for 5 minutes, a low surface coverage was observed (GT platelets, mean +/- SEM, 37.5% +/- 5.0%; normal platelets preincubated with an RGD-containing peptide, 7.4% +/- 2.1%). When rFVIIa, together with factors X and II, was added to the perfusate, platelet deposition significantly increased (GT platelets, mean +/- SEM, 67.0% +/- 4.3%; normal platelets preincubated with an RGD-containing peptide, 48.2% +/- 2.9%). The same effect was observed when normal platelets were pretreated with the commercially available anti-alpha IIb beta 3 drugs abciximab, eptifibatide, or tirofiban. It was shown that tissue factor-independent thrombin generation (presumably induced by binding of rFVIIa to adhered platelets) was responsible for the increase in platelet deposition. In conclusion, defective adhesion of alpha IIb beta 3-deficient platelets to ECM can be restored by tissue factor-independent rFVIIa-mediated thrombin formation. The enhanced generation of platelet procoagulant surface facilitates fibrin formation, so that lack of platelet aggregate formation might be compensated for.

A new approach to antiplatelet therapy: inhibitor of GPIb/V/IX-vWF interaction.

Evidence has been presented that the interaction between von Willebrand factor (vWF) and its platelet membrane receptor, the GPIb/V/IX complex, plays an important role in the pathogenesis of arterial thrombosis. A monoclonal antibody against the A1 domain of vWF has been shown to inhibit thrombus formation in the animal model of arterial thrombosis. Based upon these findings, a new approach to treating arterial thrombosis has been proposed by intervening in the interaction between vWF and platelet.

Platelet-vessel wall interactions in thrombosis and restenosis role of von Willebrand factor.

As a consequence of vessel wall injury, subendothelial matrix and collagen fibers are exposed to the flowing blood. Circulating platelets adhere to these structures and initiate arrest of blood flow. Subendothelial von Willebrand Factor (vWF) plays an important role in mediating platelet adhesion to the injured site, at least in the arterial circulation, characterized by sufficiently elevated shear forces to allow a critical conformation change in vWF, enabling an interaction between the vWF domain A1 and the vWF receptor on the platelet, the GPIb/IX complex. In vitro, in the absence of shear forces, non-physiological mediators are required to induce vWF binding to GPIb. Analysis of the mechanism according to which ristocetin induces vWF binding to GPIb revealed that 2 dimers of ristocetin simultaneously bind to vWF and GPIb, thus forming a quaternary complex in which repulsive negative charges are neutralized by the positively charged ristocetin. The interaction of vWF with its vascular receptor, i.e. collagen VI, which was isolated from human placenta and the extracellular matrix from lung fibroblasts, showed that vWF binds to collagen VI entirely via its A1 domain, i.e. via the domain that binds to GPIb. Also, vWF binding to intact extracellular matrices occurs to matrix associated collagen VI via the vWF A1 domain. By using a combination of 2 specific monoclonal anti-vWF antibodies, it was possible to induce conformational changes in WF that exposed the binding sequences in the A1 domain for GPIb. Thus, in the absence of shear forces, specific vWF binding to GPIb could be induced in the absence of any further mediators. This increased vWF binding to GPIb was sufficient to induce vWF dependent platelet aggregation, although as a consequence of Fc binding to the platelet Fc receptor, platelet activation also occurred via this pathway. Thus, general conformational changes in vWF suffice to expose the relevant amino acid sequences in the A1 domain that enable binding to GPIb. The collagen binding protein calin, isolated from the saliva of the medicinal leech, not only blocks platelet binding to collagen but also inhibited vWF binding. Thus this protein was able to inhibit both the vWF independent and vWF dependent platelet adhesion to various collagens, but much less the platelet binding to endothelial extracellular matrices, that contain matrix anchored vWF. In vivo anti-thrombotic studies in the hamster showed that the vWF antagonist aurin tricarboxylc acid was a more potent inhibitor of arterial thrombosis than of venous thrombosis, confirming the in vivo role of vWF during thrombus formation. Following vessel wall damage and thrombus formation, the neointima that formed in the hamster carotid artery developed more rapidly than in other models, and its formation partially responded to reported inhibitors of restenosis. The combination of cardiovascular drugs with complementary modes of action, such as G4120 (inhibitor of platelet GPIIb/IIIa and smooth muscle cell alpha(v) beta(3)) and quinapril (potent vascular ACE inhibitor) prevented neointima formation to about 70%, i.e. better than with any treatment separately.

Dose response of dietary fish oil supplementations on platelet adhesion.

A dose-response study of dietary fish oil supplementation on platelet adhesion was performed in three groups of five normal individuals each. Fish oil equivalent to 3, 6, or 9 g eicosapentaenoic acid (EPA)/day was administered for 3 weeks, and platelet adhesion was evaluated under high and low shear rate conditions in a laminar flow chamber before, during, and after termination of fish oil administration. Platelet adhesion to collagen I and fibrinogen, the two test surfaces in this study, was greatly reduced in response to fish oil. The inhibitory effect was similar whether platelet adhesion was evaluated at high or low shear rates. Maximal inhibitory activity was noted at 6 g EPA/day. A delayed onset and prolonged washout period characterized the response. The washout period of the fish oil effect was inversely related to the level of dietary supplementation. Measurement of total fatty acid distribution in platelets showed a dose-related increase in n-3 polyunsaturated fatty acids. From these studies, it is concluded that fish oil is an effective inhibitor of platelet adhesion, which reaches its maximum effect at approximately 6 g EPA/day.