Platelet Thrombus Volume Research Articles

Variable effect of P2Y12 inhibition on platelet thrombus volume in flowing blood

Patients treated with percutaneous coronary intervention receive aspirin and P2Y12 ADP receptor inhibitors to reduce thrombotic complications. The choice of methodology for monitoring the effects of treatment and assessing its efficacy is still a topic of debate. We evaluated how decreased P2Y12 function influences platelet aggregate (thrombus) size measured ex vivo. We used confocal videomicroscopy to measure in real time the volume of platelet thrombi forming upon blood perfusion over fibrillar collagen type I at a wall shear rate of 1500 s(-1). The average volume was significantly smaller in 31 patients receiving aspirin and clopidogrel (19) or ticlopidine (12) than in 21 controls, but individual values were above the lower limit of the normal distribution, albeit mostly within the lower quartile, in 61.3% of cases. Disaggregation of platelet thrombi at later perfusion times occurred frequently in the patients. Vasodilator-stimulated phosphoprotein phosphorylation, reflecting P2Y12 inhibition, was also decreased in the patient group, and only 22.6% of individual values were above the lower normal limit. We found no correlation between volume of thrombus formed on collagen fibrils and level of P2Y12 inhibition, suggesting that additional and individually variable factors can influence the inhibitory effect of treatment on platelet function. Measurements of platelet thrombus formation in flowing blood reflects the consequences of antiplatelet therapy in a manner that is not proportional to P2Y12 inhibition. Combining the results of the two assays may improve the assessment of thrombotic risk.

Adding Complexity to Fibronectin-Platelet Interactions

The article by Chauhan et al in the current issue of Arteriosclerosis, Thrombosis, and Vascular Biology 1 adds a new complexity to the roles of fibronectin in formation of hemostatic plugs and thrombotic occlusions. Normal mice, in which >99.5% of plasma fibronectin lacks the alternatively spliced EDA domain, were compared with mice genetically engineered to have only EDA-containing (EDA+) fibronectin in the circulation. Mice with only EDA+ fibronectin developed occlusive platelet thrombi more quickly in arterioles damaged by ferric chloride and suffered increased mortality from pulmonary emboli after an infusion of collagen and epinephrine in the tail vein. When blood from the 2 types of mice was passed through a collagen-coated chamber, surface coverage by adherent platelets was greater in the mice with EDA+ fibronectin. Examination of the photomicrographs suggests that the volume of platelet thrombi in the collagen-coated chamber also was greater after perfusion of blood from mice with EDA+ fibronectin. Three different read-outs, therefore, indicate that thrombus build-up is enhanced by the presence of EDA+ fibronectin, See …

Elevated Threshold Shear Rate for the Dependence of Glycoprotein Ibα-Mediated Platelet Thrombus Formation onto Immobilized von Willebrand Factor in Mouse Blood.

The interaction between platelet glycoprotein (GP) Ibα and von Willebrand Factor (VWF) is essential to initiate platelet deposition at sites of vascular injury and sustain platelet thrombus formation when the shear rate exceeds a threshold value. With human blood, the dependence of normal platelet adhesion and aggregation on VWF-GP Ibα function becomes evident at shear rates above 1,000 s−1. In the last several years, mouse models have been increasingly used to study the mechanisms of thrombus formation in circulating blood, and mice deficient in both VWF and GP Ibα have been generated. These animals offer the opportunity to evaluate whether the pathways of platelet adhesion and aggregation mediated by VWF and GP Ibα are equally important in mouse and human blood as well as to define the threshold shear rate at which the function of these pathways may become essential in the mouse circulation. To address this issue, we used an ex vivo perfusion system using fibrillar collagen type I as the thrombogenic surface and a flow chamber in which the shear rate varied according to a predictable function from the inlet to the outlet in relation to the x,y position in the flow path. Thus, wall shear rates between 5,000 at the inlet and 0 s−1 at the outlet could be evaluated in a single experiment, allowing a precise definition of the threshold at which platelet deposition on the surface could initiate. In these studies we used wild type control animals (WT), mice deficient in VWF (VWF-KO) and mice in which most of the extracellular domain of GP Ibα was replaced by a domain of the human interleukin 4 receptor (GPIb-KO/IL-4R). In the latter case, the ligand binding function of GP Ibα was obliterated, but unlike in GP Ib-KO mice platelet morphology and count were essentially normal. Blood was obtained from the retroorbital vein plexus and contained 100 u/ml heparin as an anticoagulant. Experiments were recorded in real time for the visualization of platelet-surface contacts and confocal videomicroscopy was used for the direct measurement of platelet thrombus volume. With normal mouse blood, platelet formed large thrombi throughout the tested range of shear rates. In contrast, with VWF-KO and GPIb-KO/IL-4R blood, thrombus volume was less than 5% of normal at 5,000 s−1, approximately 50% of normal at 3,000 s−1, but entirely normal at 1,500 s−1. Essentially the same results were observed when the extracellular matrix of mouse fibroblasts, which may better represent the complex thrombogenic properties of the vascular wall, was used as a reactive substrate instead of isolated collagen type I. The different threshold shear rate at which VWF and GP Ibα function are essential for thrombus formation with human and mouse platelets may be explained by the smaller size of the latter, which consequently are subjected to a lower drag at equivalent shear rate levels. Moreover, the similar behavior of VWF-KO and GPIb-KO/IL-4R platelets suggests that, under the conditions of these studies, VWF binding is the predominant GP Ibα function required for normal platelet thrombus formation at high shear rates. The present results should allow a more critical evaluation of the findings derived from mouse models of hemostasis and thrombosis.

Antithrombotic efficacy of inactivated active site recombinant factor VIIa is shear dependent in human blood.

Several studies have indicated a profound role for factor VII(a) [FVII(a)] in venous and arterial thrombogenesis. In the present study, we quantified the inhibitory efficacy of dansyl-glutamyl-glycyl-arginyl-recombinant FVIIa (DEGR-rFVIIa) on acute thrombus formation. Thrombus formation was elicited by immobilized tissue factor (TF) in a parallel-plate perfusion chamber device at blood flow conditions characterized by wall shear rates of 100 S-1 (veins) and 650 S-1 (medium-sized healthy arteries). Native human blood was drawn directly from an antecubital vein by a pump into a heparin-coated mixing device in which DEGR-rFVIIa (0.09 to 880 nmol/L final plasma concentration) or buffer was mixed homogeneously with flowing blood. Subsequently, the blood was passed over a plastic coverslip coated with TF and phospholipids in the parallel-plate perfusion chamber. Fibrin deposition, platelet-fibrin adhesion, and platelet thrombus volume triggered by this surface were measured by morphometry. DEGR-rFVIIa inhibited thrombus formation in a dose-dependent manner, but the efficacy was shear rate dependent. At a wall shear rate of 100 S-1, the IC50 (50% inhibition) was 30 nmol/L, whereas at 650 S-1, the IC50 was 0.6 nmol/L. Binding studies to immobilized TF under flow conditions using surface plasmon resonance revealed a significantly higher on-rate for DEGR-rFVIIa and FVIIa than for FVII, 2.8 x 10(5), 2.6 x 10(5), and 1.8 x 10(5) M-1 S-1, respectively. This indicates that a contributing factor to the shear-dependent efficacy may be a differential importance of on-rates at arterial and venous blood flow conditions.

Shear-induced platelet activation and platelet microparticle formation at blood flow conditions as in arteries with a severe stenosis.

In the present study, we investigated whether high arterial shear stresses at various exposure times or a sudden increase in shear stress introduced by a stenosis affect platelet activation and platelet microparticle formation in native human blood. We used a parallel-plate perfusion chamber device through which nonanticoagulated human blood was drawn (10 mL/min) by a pump directly from an antecubital vein through the flow channel of a perfusion chamber at wall shear rates of 420, 2600, and 10500 s-1. In another set of experiments, an eccentric stenosis was introduced into the flow channel. Wall shear rates of 2600 or 10500 s-1 at the stenosis apex were maintained at the same flow rate. The wall shear rate upstream and downstream of these stenoses was 420 s-1. A shear rate of 420 s-1 is within the range of those encountered in healthy small coronary arteries, whereas those of 2600 and 10500 s-1 are representative for vessels with various degrees of stenotic lesions. The blood was exposed to these shear rates for periods varying from 0.075 to 3.045 seconds. Platelet activation was assessed as activated glycoprotein (GP) IIb/IIIa by FITC-labeled monoclonal antibody (MAb) PAC-1 and aminophospholipid translocation by FITC-labeled annexin V. Microparticle formation was quantified by FITC-labeled MAb Y2/51 directed against GP IIIa. Significant platelet activation and formation of microparticles were observed at 10500 s-1 only (P < .008). This shear-induced platelet activation and microparticle formation were enhanced by introduction of a thrombus-promoting surface consisting of type III human collagen fibrils. Introduction of the most severe stenosis at 10500 s-1 further increased platelet activation (P < .017). The collagen-induced thrombus formation increased the platelet thrombus volume at 10500 s-1 from 16.5 to 33.8 microns3/microns2 (P < .003) on the stenosis apex when the most severe stenosis was used. A correlation (P < .0001) between platelet thrombus volume and platelet microparticle formation was observed in the presence of the eccentric stenoses. Apparently, high shear stress (315 dynes/cm2 at 10500 s-1), as encountered in severe atherosclerotic arteries, activated platelets and triggered platelet microparticle formation. In contrast, no significant platelet activation or formation of platelet microparticles was observed at physiological shear (420 s-1) or at the shear condition simulating shear in arteries with a less severe stenosis (2600 s-1). The data imply that platelets are activated and form microparticles in native blood at very high shear stresses. These events are potentiated by prolonged exposure to the high shear or by a sudden change of increasing shear due to the stenosis. The latter situation apparently enhances platelet thrombus formation at the stenosis.

Collagen Induced Thrombus Formation at the Apex of Eccentric Stenoses - A Time Course Study with Non-Anticoagulated Human Blood

Atherosclerotic plaque rupture may trigger the formation of mural thrombus. This thrombus formation is apparently affected by very high and complex shear conditions introduced by the luminal narrowing (stenosis) of the atheroma. To study the impact of such blood flow behaviour on thrombus formation we employed a model system where collagen-induced thrombogenesis is studied at the apex of well-defined eccentric stenoses. Thrombus formation in non-anticoagulated human blood drawn directly from an antecubital vein over the collagen coated stenosis apex for periods of 0.5, 1, 3 or 5 min was quantified by morphometry. The stenoses reduced the cross-sectional area of the blood flow channel by 60, 80 and 89%, which corresponded to apex wall shear rates of 2600, 10,500 and 32,000 s-1, respectively. Platelet-collagen adhesion decreased by increasing shear at the stenosis apex. The corresponding adhesion rates were highest at 1 min, then they gradually decreased upon prolongation of the perfusion time. The platelet thrombus volume increased in concert with increasing shear rate up to 10,500 s-1, whereas, at 32,000 s-1, the volume wa decreased. The corresponding growth rates and rates of thrombus occlusion at the apex levelled off at 3 min. Significant fibrin deposition was not observed before 3 min, and was most pronounced at 10,500 and 32,000 s-1. The plasma levels of fibrinopeptide A and beta-thromboglobulin increased in concert with increasing shear and perfusion time, particularly at the two highest shear conditions. Thus, hallmarks of thrombus formation at these stenoses with increasing shear are decreased platelet-collagen adhesion, and increased platelet-platelet interaction and fibrin deposition. A fibrin tail downstream to the collagen-attached platelet thrombus is regularly observed when thrombus occlusion exceeds 40%. However, the reduced thrombus growth at the most occlusive stenosis (89%) is presumably due to the high shear stresses which may reduce the rate of platelet incorporation into the thrombus and/or tear off thrombus fragments.

Reduced Effect of Aspirin on Thrombus Formation at High Shear and Disturbed Laminar Blood Flow

Aspirin is the most commonly used antithrombotic drug in primary and secondary prophylaxis against cardio- and cerebrovascular disease. In previous studies from our laboratory it was demonstrated that the effect of aspirin on collagen-induced thrombus formation in a parallel- plate perfusion device with laminar blood flow is shear rate dependent. Although aspirin did not affect collagen-induced thrombus formation at 650 s-1 (medium sized arteries), a significant inhibition of thrombus formation by approximately 38% at 2,600 s-1 (moderately stenoses in medium sized arteries) was observed. At present we have extended these studies to thrombus formation at the apex of eccentric stenoses in a parallel-plate perfusion chamber device. The stenoses reduced the cross-sectional area of the blood flow channel of the perfusion chambers by 60 or 80%, introducing disturbed laminar flow and apex wall shear rates of 2,600 and 10,500 s-1, respectively. The corresponding wall shear stresses were 80 and 315 dynes/cm2, respectively. Aspirin reduced the platelet thrombus volume at the 60% stenosis by 45% (p < 0.03), and the fibrin deposition by 70% (p < 0.004). However, none of these parameters were affected by aspirin at the 80% stenosis. These observations may at least partly explain why aspirin has a limited clinical effect in preventing arterial thrombus formation in atherosclerotic vessels at high shear and disturbed blood flow. In contrast, thrombus formation in blood from one patient with Glanzmann's thrombasthenia and two patients with von Willebrand disease subtype 2M was almost abolished at this blood flow condition. Thus, blocking the function of either von Willebrand factor or glycoprotein IIb/IIIa may represent better antithrombotic approaches for such critical events than blocking the prostaglandin metabolism by aspirin. The lack of effect of aspirin on thrombus formation at the 80% stenosis may reflect shear-induced platelet activation at the stenosis inlet region, since shear-induced platelet aggregation in rotational viscometers is not affected by aspirin at shear stresses exceeding 100 dynes/cm2.

Retinoic Acid Reduces Induction of Monocyte Tissue Factor and Tissue Factor/Factor Vila-Dependent Arterial Thrombus Formation

Agents that downregulate the induction of monocyte/macrophage tissue factor (TF) activity may attenuate the thrombotic risk associated with mechanical restoration of vessel patency or artificial arterial grafting. In such events, procoagulant macrophages in the atherosclerotic plaque and procoagulant monocytes adherent to artificial materials may be exposed to the blood stream. Ishii et al (Blood 80:2556, 1992) reported that induction of endothelial TF is downregulated by all-trans retinoic acid (ATRA), and Conese et al (Thromb Haemost 66:662, 1991) reported that retinoids downregulate monocyte procoagulant activity (PCA). These findings led us to investigate the effect of ATRA on monocyte TF expression, and to study the effect of ATRA on monocyte-induced thrombus formation in a model system of human arterial thrombogenesis. Induction of PCA in human peripheral blood monocytes by 0.5 microgram/mL lipopolysaccharide (LPS) was dose dependently reduced by ATRA, reaching a reduction of 56% at 10(-5) mol/L ATRA (P < .0001). A 38% reduction (P < .0007) in LPS-induced TF antigen expression was observed at an ATRA concentration of 10(-6) mol/L. Adherence of monocytes to plastic cover slips (Thermanox, Miles Laboratories, Naperville, IL) also triggered induction of cellular PCA, which was inhibited by more than 80% by an anti-TF monoclonal antibody (MoAb) (P < .002). Inclusion of ATRA (10(-6) mol/L) reduced this PCA by 40% (P < .03), and the TF antigen expression by 30% (P < .0001). Exposure of Thermanox adherent monocytes to flowing nonanticoagulated human blood in a parallel-plate perfusion chamber device at an arterial wall shear rate of 650 s-1 elicited significant fibrin deposition and platelet thrombus formation. Partial interruption of this thrombus formation was achieved by 10(-6) mol/L ATRA, which reduced the fibrin deposition by 80% (P < .02) and platelet thrombus formation by 50% (P < .05). In comparison, incubation of adherent monocytes with the anti-TF MoAb before the blood exposure, reduced the fibrin deposition by 83% (P < .02) and platelet thrombus volume by 75% (P < .0008). Thus, ATRA is an effective down-regulator of monocyte TF-PCA, and may reduce thrombotic complications at sites of plaque rupture, at plaque disruption after percutaneous transluminal angioplasty procedures, or on surfaces introduced by artificial arterial grafting.

Procoagulant human monocytes mediate tissue factor/factor VIIa-dependent platelet-thrombus formation when exposed to flowing nonanticoagulated human blood.

Tissue factor (TF) on monocyte and macrophage surfaces is a nonproteolytic cofactor for factor VIIa (FVIIa)-induced coagulation. Monocyte-derived macrophages in atherosclerotic plaques express TF, which, after plaque disruption or rupture, may complex with FVII/VIIa from the bloodstream, resulting in activation of extrinsic coagulation. We studied the effect of TF expression on human monocytes on arterial thrombus formation in a model system of thrombogenesis. Thawed, cryopreserved human monocytes adherent to plastic coverslips were stimulated with lipopolysaccharide (0.5 microgram/mL) to express TF and subsequently exposed to flowing nonanticoagulated human blood in a parallel-plate perfusion chamber. The wall shear rate at the cell surface was 650 seconds-1, corresponding to that of average-sized coronary arteries. The stimulated monocytes elicited pronounced fibrin deposition and platelet-thrombus formation. The platelet-thrombus volume was as large as that triggered by human type III collagen fibrils under similar experimental conditions. In contrast, the monocytes elicited much more fibrin deposition than the collagen surface. However, inclusion of an anti-TF monoclonal antibody that blocks the complexation of FVII/FVIIa with TF virtually abolished the fibrin deposition (P < .03) and reduced platelet-thrombus formation by more than 70% (P < .04). Thus, arterial thrombus formation induced by stimulated monocytes was almost completely blocked by the anti-TF antibody, suggesting that inhibition of TF/FVIIa complex formation on monocytes and macrophages at sites of plaque rupture or after percutaneous transluminal coronary angioplasty procedures may reduce intravascular thrombotic complications.

Dose- and shear rate-dependent effects of heparin on thrombogenesis induced by rabbit aorta subendothelium exposed to flowing human blood.

The effect of heparin on thrombogenesis induced by the subendothelium of rabbit aorta was investigated in 24 healthy volunteers after intravenous injection of different doses (0, 1000, 2500, and 5000 IU). By using an ex vivo perfusion chamber system, the interaction between flowing blood and exposed subendothelium was measured at low (50 s-1), intermediate (650 s-1), and high (2600 s-1) wall shear rates. The low shear rate simulated blood flow in venous, the intermediate shear rate in arterial, and the high shear rate in small or stenosed arterial vessels. Deposition of fibrin, platelets, and platelet thrombi on vascular subendothelium (SE) was quantified by morphometrical and immunological techniques. Fibrin deposition prevailed at low shear rates and was only minimal at high shear rates (30 +/- 1% vs. 1 +/- 0.4% coverage of SE with fibrin, means +/- SEM, p less than 0.001). In contrast, the interaction of platelets with SE was more intense at high compared to low shear rates, as indicated by higher platelet adhesion (54 +/- 5% vs. 4 +/- 1% coverage of SE with platelets, p less than 0.001) and platelet thrombus volumes (4.8 +/- 1.3 vs. 0.5 +/- 0.1 microns 3/microns 2, p less than 0.001). Fibrin deposition on SE was inhibited by heparin in a dose-dependent manner and was abolished after high doses. In addition, high doses of heparin reduced the height and volume of platelet thrombi at low and intermediate wall shear rates, but no effect was found at the high shear rate. Our data show that heparin inhibits the formation of both fibrin and platelet thrombi on vascular subendothelium. The lack of effect of heparin on platelet thrombus formation at high shear rates indicates that thrombin modulates the growth rate and/or stability of platelet thrombi at low and intermediate shear rates, whereas additional factors may control platelet thrombus growth and stability at high shear conditions.

Effects of red blood cell concentration on hemostasis and thrombus formation in a primate model

Because the effects of red blood cell (RBC) concentration on hemostasis and thrombus formation have not been studied experimentally under conditions of whole blood flow without anti-coagulation, normal baboons were bled or transfused to obtain three different groups: a low hematocrit (Ht) group (20% less than Ht less than 25%), a normal Ht group (35% less than Ht less than 40%), and a high Ht group (50% less than Ht less than 55%). Measurements of platelet count, bleeding time, platelet aggregation, fibrinogen level, and coagulation time (APTT) were equivalent to normal values in each group. Thrombus formation was induced using a device composed of collagen-coated tubing followed by two sequentially placed expansion chambers designed to exhibit flow recirculation and stasis. The device was exposed for up to 40 minutes in an arterio-venous shunt system. Wall shear rates in the tubular collagen segment were 100 seconds-1 and 500 to 750 seconds-1. The accumulation of 111In-platelets and 125I-fibrinogen/fibrin was measured radioisotopically; RBC incorporation was determined from measurements of total thrombus hemoglobin. Thrombus that formed on the collagen substrate was rich in platelets and poor in fibrin and RBCs. Under high flow conditions, thrombus composition showed no dependence on Ht. Surprisingly, under low flow conditions, platelet thrombus volume was negatively correlated with Ht (r = -.73, P = .005), and was increased by greater than twofold in the low Ht group as compared with the high Ht group. Thrombus that formed in the disturbed flow regions contained relatively few platelets but was rich in fibrin and RBCs. The predominant finding was a positive correlation between RBC incorporation and Ht at both high and low shear rates (r = .90, P = .00003; and r = .77, P = .002, respectively), with thrombus volume increasing three- to sixfold between the low and high Ht groups. Thus, in vivo variations in Ht ranging between 20% and 55% did not affect hemostasis, but were found either to promote or inhibit the net accumulation of thrombus, depending on local flow conditions.

Effects of Red Blood Cell Concentration on Hemostasis and Thrombus Formation in a Primate Model

Because the effects of red blood cell (RBC) concentration on hemostasis and thrombus formation have not been studied experimentally under conditions of whole blood flow without anti-coagulation, normal baboons were bled or transfused to obtain three different groups: a low hematocrit (Ht) group (20% less than Ht less than 25%), a normal Ht group (35% less than Ht less than 40%), and a high Ht group (50% less than Ht less than 55%). Measurements of platelet count, bleeding time, platelet aggregation, fibrinogen level, and coagulation time (APTT) were equivalent to normal values in each group. Thrombus formation was induced using a device composed of collagen-coated tubing followed by two sequentially placed expansion chambers designed to exhibit flow recirculation and stasis. The device was exposed for up to 40 minutes in an arterio-venous shunt system. Wall shear rates in the tubular collagen segment were 100 seconds-1 and 500 to 750 seconds-1. The accumulation of 111In-platelets and 125I-fibrinogen/fibrin was measured radioisotopically; RBC incorporation was determined from measurements of total thrombus hemoglobin. Thrombus that formed on the collagen substrate was rich in platelets and poor in fibrin and RBCs. Under high flow conditions, thrombus composition showed no dependence on Ht. Surprisingly, under low flow conditions, platelet thrombus volume was negatively correlated with Ht (r = -.73, P = .005), and was increased by greater than twofold in the low Ht group as compared with the high Ht group. Thrombus that formed in the disturbed flow regions contained relatively few platelets but was rich in fibrin and RBCs. The predominant finding was a positive correlation between RBC incorporation and Ht at both high and low shear rates (r = .90, P = .00003; and r = .77, P = .002, respectively), with thrombus volume increasing three- to sixfold between the low and high Ht groups. Thus, in vivo variations in Ht ranging between 20% and 55% did not affect hemostasis, but were found either to promote or inhibit the net accumulation of thrombus, depending on local flow conditions.