A New Method for Quantifying Platelet Deposition in Flowing Native Blood in an Ex Vivo Model of Human Thrombogenesis

Abstract

SummaryNo quantitative, simple and non-radioactive method has been described for measuring the platelet content of experimental thrombi. The aim of the present study was to develop a simple method for quantifying platelets in thrombi formed on thrombogenic surfaces in flowing native human blood. To test the relevance of this new method, the effect of unfractionated heparin on arterial thrombus formation was investigated. Tissue factor (TF)- and collagen-coated coverslips were exposed to non-anticoagulated blood at an arterial wall shear rate (2,600 s–1) for 1 to 4 min. Platelet deposition was quantified by measuring the P-selectin (PS) and β-thromboglobulin (βTG) content of dissolved plasmin-digested thrombi using immunoenzymoassays; fibrin deposition was determined by measuring the D-dimer levels. These results were compared to those established by morphometrical analysis.Morphometric evaluation showed that fibrin deposition was maximum on TF by 1 min perfusion time. Platelets deposited subsequently and reached a maximum at 3 min. On collagen, platelets deposited directly on the collagen fibrils without detectable fibrin deposit. Platelet deposition increased from 1 to 4 min. Platelet deposition quantified by PS was correlated to the values obtained by morphometry (r = 0.72, r = 0.67, p <0.001, on TF and collagen, respectively). As compared to PS, βTG measurements gave an underestimation of the size of the thrombus platelet number. Unfractionated heparin infused through a mixing device proximal to the perfusion chamber to obtain plasma concentrations of 0.5, 1 and 3 IU/ml, reduced fibrin deposition on TF-coated coverslips in a dose-dependent manner (77% reduction at 3 IU/ml, p <0.01), but had no significant effect on platelet deposition (33% at 3 IU/ml, p >0.05). In contrast, heparin had no effect on fibrin or platelet deposition on collagen-coated coverslips.Thus, a new quantitative and simple method for measuring platelet deposition in flowing blood has been developed and characterized. Utilizing this system, we have demonstrated that unfractionated heparin did not inhibit arterial thrombus formation either on procoagulant or on proaggregant surface.

Antithrombotic Efficacy of the Vitamin K Antagonist Fluindione in a Human Ex Vivo Model of Arterial Thrombosis

“…The efficacy of antithrombotic drugs is determined by quantification of the respective thrombus content in platelets and fibrin by immunoenzymatic methods. 17,20 This model has been used to investigate numerous antithrombotic strategies, and results appear consistent with clinical data. 17,[21][22][23][24][25][26][27][28][29] In the present study, we used this ex vivo model of acute initial arterial thrombus formation to determine the efficacy of the vitamin K antagonist fluindione in preventing arterial thrombosis, the level of anticoagulation required to obtain an antithrombotic effect, and whether fluindione in combination with aspirin improves the antithrombotic efficacy of fluindione alone.…”

“…[13][14][15][16][17] In this model, native blood is drawn from volunteers through a parallel-plate chamber device, where it interacts with a thrombogenic surface in well-established blood flow conditions, mimicking wall shear rates encountered in moderately stenosed arteries (2600 s Ϫ1 ). Two relevant thrombogenic molecules, collagen and tissue factor (TF), which are present in atherosclerotic plaques and primarily responsible for thrombus formation in vivo, 18,19 are exposed to blood.…”

Role of 4 platelet membrane glycoprotein polymorphisms on experimental arterial thrombus formation in men

“…Subsequently, the coverslip covered by thrombotic deposits was placed in a plasmin solution and processed as previously described. 5 …”

“…5 After centrifugation of the plasmindigested thrombus, the pellet was dissolved in 400 L lytic buffer, frozen and thawed 3 times, and then sonicated (4°C, 20 KHz) for 270 seconds. All samples of dissolved pellets were stored at Ϫ80°C until assayed for P-selectin by immunoenzymoassay (Bender MedSystems, Vienna, Austria).…”

“…We used an experimental model of arterial thrombus formation device in men that closely mimics relevant clinical situations. [3][4][5][6][7] In this model, native blood is drawn from healthy volunteers through a parallel-plate chamber device where blood components interact with collagen at wellestablished flow conditions. Blood flow conditions mimic wall shear rates as encountered in medium-sized (650 s Ϫ1 ) and moderately stenosed small (2600 s Ϫ1 ) arteries.…”