Arterial Blood Flow Conditions Research Articles

Dynamic in vitro hemocompatibility of oligoproline self-assembled monolayer surfaces.

The blood compatibility of self-assembled monolayers (SAMs) of oligoproline, a nonionic antifouling peptide, was investigated using the cone-and-plate assay imitating arterial blood flow conditions. End-capped oligoprolines composed of 6 and 9 proline residues (Pro6 and Pro9) and a Cys residue were synthesized for preparing SAMs (Pro-SAMs) on Au-sputtered glass. The surface of Pro-SAMs indicated hydrophilic property with a smooth topology. The adsorption of blood components and the adhesion of blood cells, including leukocytes and platelets, were strongly suppressed on Pro-SAMs. Moreover, Pro9-SAM did not trigger the activation of platelets (i.e., the conformational change of GPIIb/IIIa and P-selectin (CD62P) expression on platelets and the formation of aggregates). Our results demonstrate that Pro9-SAM completely inhibited acute thrombogenic responses and the activation of platelets under dynamic conditions.

Pharmacological Blockade of Glycoprotein VI Promotes Thrombus Disaggregation in the Absence of Thrombin.

Atherothrombosis occurs upon rupture of an atherosclerotic plaque and leads to the formation of a mural thrombus. Computational fluid dynamics and numerical models indicated that the mechanical stress applied to a thrombus increases dramatically as a thrombus grows, and that strong inter-platelet interactions are essential to maintain its stability. We investigated whether GPVI (glycoprotein VI)-mediated platelet activation helps to maintain thrombus stability by using real-time video-microscopy. Approach and Results: We showed that GPVI blockade with 2 distinct Fab fragments promoted efficient disaggregation of human thrombi preformed on collagen or on human atherosclerotic plaque material in the absence of thrombin. ACT017-induced disaggregation was achieved under arterial blood flow conditions, and its effect increased with wall shear rate. GPVI regulated platelet activation within a growing thrombus as evidenced by the loss in thrombus contraction when GPVI was blocked, and the absence of the disaggregating effect of an anti-GPVI agent when the thrombi were fully activated with soluble agonists. The GPVI-dependent thrombus stabilizing effect was further supported by the fact that inhibition of any of the 4 key immunoreceptor tyrosine-based motif signalling molecules, src-kinases, Syk, PI3Kβ, or phospholipase C, resulted in kinetics of thrombus disaggregation similar to ACT017. The absence of ACT017-induced disaggregation of thrombi from 2 afibrinogenemic patients suggests that the role of GPVI requires interaction with fibrinogen. Finally, platelet disaggregation of fibrin-rich thrombi was also promoted by ACT017 in combination with r-tPA (recombinant tissue plasminogen activator). This work identifies an unrecognized role for GPVI in maintaining thrombus stability and suggests that targeting GPVI could dissolve platelet aggregates with a poor fibrin content.

The impact of factor Xa inhibition on axial dependent arterial thrombus formation triggered by a tissue factor rich surface

This study was designed to assess the effect of Factor Xa antagonists on thrombus formation at various axial positions on a tissue factor rich surface under arterial blood flow conditions. Non-anticoagulated, flowing human blood, drawn directly from an antecubital vein, was perfused over a tissue factor coated cover slip in a parallel-plate perfusion chamber. Thrombus surface coverage, thrombus mean height and fibrin surface coverage were measured at six different axial positions by confocal microscopy. Both thrombus surface coverage and mean height decreased along the cover slip axis whereas the fibrin surface coverage increased. Pre-chamber treatment of blood with the direct Factor Xa inhibitors Razaxaban and 813893 resulted in significantly reduced thrombus and fibrin formation at all axial positions investigated (P<0.05). Thrombus and fibrin deposition in a laminar flow chamber changed with axial position with surface coverage measurements being more reproducible than thrombus mean height. Data were more reproducible towards the centre of the flow chamber than at the extremities. Razaxaban and 813893 inhibited thrombus and fibrin formation at the highest concentrations tested. No difference in drug effect was apparent at different axial positions. In conclusion, axial position influences the degree of thrombus and fibrin deposition with measurements being less reproducible at the extremities of the flow chamber. This technique may prove useful for analysing anti-thrombotic drug effects before progression to clinical trials.

Analysis of mechanisms for platelet near-wall excess under arterial blood flow conditions

The concentration of platelets near the blood vessel wall is important because platelets survey the condition of the vessel wall and respond to injuries to it. Under arterial flow conditions, platelets are non-uniformly distributed across the vessel lumen and have a high concentration within a few microns of the vessel wall. This is believed to be a consequence of the complex motion of red blood cells which constitute a large fraction of the blood's volume. We use a novel lattice Boltzmann-immersed boundary method to simulate, in two dimensions, the motion of dense red blood cell suspensions and their effect on platelet-sized particles. We track the development of a red blood cell-free layer near the wall and the later development of the platelet near-wall excess. We find that the latter develops more quickly at high wall shear rates and that the magnitude of the excess and its proximity to the wall are dependent on haematocrit. Treating the simulation data as if it were generated by a drift–diffusion process, we find that the effective lateral platelet diffusivity depends strongly on lateral position; it has a magnitude of order of 10−6 cm2 s−1 over much of the lumen but drops to almost zero close to the wall. This large effective diffusivity over the core of the lumen combined with reduced space for platelets in this region because of the inward migration of red blood cells largely but not completely accounts for the observed platelet-concentration profiles. We present evidence for a highly localized red blood cell-induced platelet drift at the edge of the red cell-free layer and suggest a physical mechanism that may generate it.

Important Regulatory Role of Activated Platelet-Derived Procoagulant Activity in the Propagation of Thrombi Formed Under Arterial Blood Flow Conditions

The functional links between the activation of platelets and the coagulation system have not been clarified. Immobilized collagen fibrils were perfused with human blood containing fluoresceinated platelets in the presence of various concentrations of thrombin inhibitor. Coagulant activity around platelet thrombi was detected using a FITC-conjugated antibody against the fibrin monomer complex (F-405). Intra-cytosolic calcium ion concentrations ([Ca(2+)](i)) in individual platelets and the volume of thrombi were detected with an ultrafast confocal laser microscope equipped with a piezo-motor control unit. The volume of platelet thrombi formed after 8 min of blood perfusion in the presence of 10, 25, 50, and 100 micromol/L argatroban was 7.69+/-0.46 microm(3), 6.61+/-1.96 microm(3), 3.63+/-1.54 microm(3), and 1.67+/-0.75 microm(3), respectively. There was a positive correlation between the volume of platelet thrombi and the amount of fibrin monomer complex produced around them. The [Ca(2+)](i) of the platelets forming the thrombi oscillated between a minimum of 92.0+/-57.4 nmol/L, 120.1+/-68.1 nmol/L, and a maximum of 217.6+/-131.5 nmol/L, 367.6+/-189.1 nmol/L, respectively, in the presence of 100 and 10 mumol/L argatroban. The results suggest a crucial role of coagulant activity in both the generation of fibrin and the growth of platelet thrombi.

Propagation of Arterial Thrombi

It is a common understanding that the rupture of an atheroma in the coronary arteries is the initial event in the onset of arterial thrombosis resulting in myocardial infarction.1 Ex vivo perfusion experiments using human blood have clearly demonstrated that platelet accumulation occurs immediately when the subendothelial matrix, such as collagen, is exposed to the blood stream.2 However, initiation of platelet thrombus formation after endothelial disruption resulting in exposure of the subendothelial matrix does not directly represent the onset of symptomatic atherothrombotic diseases, such as myocardial infarction, which were caused by thrombotic arterial occlusion. For example, coronary intervention, while causing damage to the endothelium, does not, in most cases, result in any symptomatic myocardial ischemia. Moreover, recent advances in clinical imaging techniques, such as intracoronary ultrasonography, have revealed a much higher incidence of atheroma rupture than of symptomatic atherothrombotic coronary artery diseases, including myocardial infarction and unstable angina pectoris.3,4 These observations suggest the contribution of propagating factors for thrombus growth, besides exposure of the subendothelial matrix due to endothelial disruption, in the onset of symptomatic arterial thrombotic diseases. See …

Inhalation of nitric oxide inhibits ADP-induced platelet aggregation and alpha-granule release

To gather further information about the effects on blood platelet activation of in vivo exposure to nitric oxide (NO), platelet reactivity was studied in blood from healthy, non-smoking male volunteers before and after 30 min inhalation of 40ppm NO. Whole blood was stimulated in vitro with adenosine diphosphate or thrombin receptor activation peptide (TRAP-6). In an ex vivo perfusion model, non-anticoagulated blood was exposed to immobilised collagen at arterial blood flow conditions (2600 s -1 ). Blood samples from both the in vitro and ex vivo experiments were stained with fluorochrome-labelled Annexin-V and antibodies against CD42a, CD45, CD49b, CD61, CD62P and fibrinogen, and analysed with a three-colour flow cytometry technique. NO inhalation reduced the platelet activation response to adenosine diphosphate (ADP) stimulation by decreasing platelet-platelet aggregation, alpha-granule release and platelet-leukocyte conjugate formation. TRAP-stimulated platelet activation, collagen-induced platelet activation and thrombus growth was unaffected by NO inhalation. We therefore suggest an ADP receptor inhibitor mode of action of inhaled NO, selective on the newly suggested G protein- and phospholipase C-coupled P2Y1 receptor. Our results demonstrate that blood platelet activation in healthy subjects is modulated by inhalation of NO in therapeutically relevant doses, although the clinical impact of our findings remains unclear.

ENDOGENOUS NITRIC OXIDE RELEASE MODULATES MURAL PLATELET THROMBOSIS AND NEUTROPHIL-ENDOTHELIUM INTERACTIONS UNDER LOW AND HIGH SHEAR CONDITIONS

Nitric oxide (NO) plays an important role in the maintenance of a constant vasodilator tone in the vasculature and confers anti-adhesive properties to the normal functioning endothelium. Whether endogenous NO release influences platelet thrombus formation and neutrophil-endothelium interactions under arterial blood flow conditions was investigated in ex vivo bioassay experiments using superfusion flow chambers. Surfaces of intact or deeply injured porcine arterial segments were exposed to flowing porcine arterial blood under shear conditions typical to patent (424 sec −1) and stenosed (3397 sec −1) arteries, at baseline and after administration of the specific inhibitor of NO synthesis N ω-nitro- l-arginine methyl ester ( l-NAME, 3 mg/kg + 3 mg/kg/h; i.v.). l-NAME induced a rapid and significant rise in arterial blood pressure, with a moderate reduction in heart rate. 51Cr platelet deposition on the exposed arterial media, which averaged 15.9±2.9×10 6/cm 2 at a shear rate of 424 sec −1, was increased by l-NAME, to 20.4±2.8×10 6/cm 2 ( p<0.05). At 3397 sec −1 of shear rate, platelet deposition was higher (71.4±11.9×10 6/cm 2) ( p<0.001), and was enhanced by 34%, to 95.8±12.5×10 6/cm 2 ( p<0.05), after l-NAME treatment. 111In neutrophil adhesion to the vascular endothelium was also increased by l-NAME by 83%, from 10.6±2.5 to 19.4±5.7×10 3/cm 2 ( p<0.05) at 424 sec −1, and by 110%, from 14.1±4.3 to 29.7±10.0×10 3/cm 2 ( p<0.05) at 3397 sec −1 of shear rate. These results suggest that endogenous NO may be an important modulator of thrombotic and inflammatory processes in patent as well as in stenosed arteries. Copyright © 1997 Elsevier Science Ltd

Tissue factor-induced coagulation triggers platelet thrombus formation as efficiently as fibrillar collagen at arterial blood flow conditions.

The relative importance of vessel wall tissue factor (TF) in initiating thrombogenesis is not well defined. In contrast, vessel wall collagens have been well documented as potent inducers of thrombus formation. We compared the potency of a human TF/phospholipid surface with that of a surface consisting of human type III collagen fibrils in triggering thrombus formation in native human blood at venous and arterial blood flow conditions. A commercial preparation, Thromborel S, was used as a source of human TF. Biochemical characterization of this preparation revealed small amounts of FVII, FIX, and FX proteins. Coagulant activity of these proteins was associated with the FVII protein only, although it was a very low activity. Studies with anti-TF antibodies in a one-stage clotting assay showed that the procoagulant activity of Thromborel was mainly a result of TF. The molar ratio of TF to phospholipid was 1:2 x 10(7). Thrombus formation in flowing nonanticoagulated human blood drawn directly from an antecubital vein was triggered by either Thromborel S or collagen fibrils coated on Thermanox coverslips in a parallel-plate perfusion chamber device. A 1:50 Thromborel S dilution gave maximal fibrin deposition (90% surface coverage) at a wall shear rate of 100 s-1. However, pretreatment of the TF surface with a monoclonal anti-TF antibody reduced this fibrin deposition by 93% (P < .001). Thus, TF was essential for the procoagulant activity of the Thromborel S surface in this flow system also. At higher wall shear rates (650 and 2600s-1), less fibrin was deposited, but the platelet thrombus formation on the fibrin mesh increased dramatically.(ABSTRACT TRUNCATED AT 250 WORDS)

Annexin V inhibits the procoagulant activity of matrices of TNF-stimulated endothelium under blood flow conditions.

A human ex vivo thrombosis model was used to investigate whether recombinant annexin V (rANV) can prevent thrombus formation under venous and arterial blood flow conditions. In this model, blood from an antecubital vein of healthy donors was allowed to flow directly over the extracellular matrix of tumor necrosis factor-stimulated endothelial cells (TNF-ECMs). TNF-ECMs were preincubated with rANV (2.9 mumol/L) for 30 minutes. With this rANV concentration all binding sites present on TNF-ECMs (1.6 +/- 0.5 x 10(12)/cm2) are occupied, and a maximal inhibition was observed in a tissue factor-dependent clotting assay. Fibrin deposition and platelet and leukocyte adhesion were measured on the rANV-treated and nontreated TNF-ECMs. Nontreated TNF-ECMs were used as controls. rANV inhibited fibrin deposition by 81% at a wall shear rate of 100 s-1. A nonsignificant inhibition was also observed at 650 s-1. Platelet-matrix adhesion, which is more prominent at higher shear rates, was significantly decreased by 60% at 100 s-1 but not at 650 s-1. The average leukocyte adherence was nonsignificantly lowered at 100 s-1. Virtually no leukocytes adhered at 650 s-1. The results demonstrated that rANV can inhibit blood coagulation under venous blood flow conditions and may serve as an antithrombotic drug.

HN-11500 – a Novel Thromboxane A2 Receptor Antagonist With Antithrombotic Activity in Humans at Arterial Blood Flow Conditions

In the present study we have investigated the effect of a 100 mg single oral dose of a newly developed thromboxane A2 receptor antagonist on collagen-induced thrombogenesis in flowing human non-anticoagulated blood. Blood was drawn directly from an antecubital vein over immobilised collagen type III fibrils on a cover slip placed in a parallel-plate perfusion chamber. Shear rates at the collagen surface were characteristic for medium sized (650 s-1) and moderately stenosed (2,600 s-1) arteries. Blood-collagen interactions were morphologically quantified as platelet-collagen adhesion, fibrin deposition and thrombus volume. Activation peptides of coagulation, fibrinopeptide A (FPA), and of platelets, beta-thromboglobulin (beta-TG), were measured immediately distal to the perfusion chamber. HN-11500 ingestion reduced significantly the thrombus volume by 32% at 2,600 s-1, but not at 650 s-1. However, transmission electron microscopy revealed loosely packed and less degranulated platelets at 650 s-1. The beta-TG plasma levels were also reduced at both shear rates by the HN-11500 ingestion. The platelet-collagen adhesion was significantly enhanced at both shear rates. This was apparently a consequence of higher platelet concentrations at the collagen surface, because fewer platelets were consumed by the thrombi after the drug ingestion. In contrast, the coagulation, as measured by fibrin deposition and FPA plasma levels, was not significantly affected by HN-11500. Thus, it appears that the thromboxane A2 receptor antagonist HN-11500 reduces the thrombotic response by primarily impairing the platelet function at arterial blood flow conditions, and particularly at high wall shear rates.

In Vivo Inhibition of Acute Platelet-Dependent Thrombosis in a Baboon Model by BAY U3405, a Thromboxane A2-Receptor Antagonist

Bay U3405 is a thromboxane A2 (TxA2)-receptor antagonist that inhibits the binding of TxA2 to its target cells. The aim of this study was to determine if Bay U3405 could be used to inhibit arterial thrombosis. A thrombogenic device, consisting of uncrimped Dacron vascular graft material (0.5 cm2) built into the wall of silicone rubber tubing with 4 mm inside diameter, was exposed to native flowing blood under arterial blood flow conditions (100-140 ml/min) by interposing the devices as extension segments into permanent femoral arteriovenous shunts implanted in baboons. Thrombus formation was quantified in vivo by measuring the deposition of 111In-labelled platelets onto the graft material with a scintillation camera. In six baboons, a bolus injection of Bay U3405, calculated to attain an initial plasma concentration of 300 ng/ml, reduced the maximum thrombus formation measured over a 2 h study period. Platelet deposition was reduced by 33 +/- 14% (SD) at 2 h as compared to control studies done in the same baboons. The accumulation of additional platelets onto a thrombus that was allowed to form for 1 h, was reduced by 58 +/- 28% at 2 h. Ex vivo platelet aggregation in response to ADP, activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) were not affected by the treatment. Ex vivo platelet aggregation in response to collagen was markedly inhibited for 2 h after treatment. The results demonstrated that selective blocking of the TxA2-receptor on platelets reduced platelet-dependent thrombus formation and the accumulation of additional platelets in a freshly formed thrombus.(ABSTRACT TRUNCATED AT 250 WORDS)

Confluent durable endothelialization of endarterectomized baboon aorta by early attachment of cultured endothelial cells

Since surgical endarterectomy produces a highly thrombogenic vascular flow surface, we evaluated in baboons the capacity of acutely attached, unspread, cultured, homologous aortic endothelial cells to form durable, confluent endothelial cell monolayers and to interrupt acute thrombus formation during exposure to arterial blood flow conditions. When cultured baboon aortic endothelial cells were incubated with segments of freshly endarterectomized baboon aorta for 20 minutes in vitro, 3.8 x 10(5) endothelial cells/cm2 became attached. The endarterectomized surface treated with endothelial cells showed the presence of rounded, unspread endothelial cells with intervening bare areas of vascular media. Platelet deposition onto the endothelial cell--treated surface during exposure to blood under high flow conditions for 1 hour in vivo was markedly reduced by the attached but unspread endothelial cells. Moreover, these attached endothelial cells underwent spreading on the endarterectomized surface in the presence of high shear blood flow to form a confluent endothelial monolayer at the end of 1 hour. Thus acutely attached cultured endothelial cells form a durable thromboresistant monolayer on surgically denuded vascular surfaces during exposure to arterial blood flow.

Confluent durable endothelialization of endarterectomized baboon aorta by early attachment of cultured endothelial cells

Since surgical endarterectomy produces a highly thrombogenic vascular flow surface, we evaluated in baboons the capacity of acutely attached, unspread, cultured, homologous aortic endothelial cells to form durable, confluent endothelial cell monolayers and to interrupt acute thrombus formation during exposure to arterial blood flow conditions. When cultured baboon aortic endothelial cells were incubated with segments of freshly endarterectomized baboon aorta for 20 minutes in vitro, 3.8 × 105 endothelial cells/cm2 became attached. The endarterectomized surface treated with endothelial cells showed the presence of rounded, unspread endothelial cells with intervening bare areas of vascular media. Platelet deposition onto the endothelial cell-treated surface during exposure to blood under high flow conditions for 1 hour in vivo was markedly reduced by the attached but unspread endothelial cells. Moreover, these attached endothelial cells underwent spreading on the endarterectomized surface in the presence of high shear blood flow to form a confluent endothelial monolayer at the end of 1 hour. Thus acutely attached cultured endothelial cells form a durable thromboresistant monolayer on surgically denuded vascular surfaces during exposure to arterial blood flow.

Platelet Adhesion and Platelet Thrombus Formation on Subendothelium of Human Arteries and Veins Exposed to Flowing Blood &lt;i&gt;in vitro&lt;/i&gt;. A Comparison with Rabbit Aorta

Subendothelium of blood vessel segments from elderly patients was exposed in a perfusion chamber to citrated blood under arterial blood flow conditions. The resulting platelet adhesion and thrombus formation was assessed morphometrically. The results show that the time course and the extent of platelet adhesion and aggregation were similar to the results obtained previously with subendothelium from rabbit arteries. The extent of surface-induced platelet aggregation was furthermore governed by both the properties of the exposed surface as well as those of the blood perfusate. No significant differences with regard to platelet reactivity were found between veins and arteries and the extent of surface-induced platelet aggregation was rather low.