Effect of Clopidogrel on Thrombus Formation in an Ex Vivo Parallel Plate Flow Chamber Model Cannot Be Reversed by Addition of Platelet Concentrates or vWF Concentrate.

BACKGROUND: Hemorrhage is the most important complication of antithrombotic therapy with P2Y12 receptor blockers. The administration of platelet concentrates (PCs) and von Willebrand factor (vWF) concentrates are common procedures to normalize impaired primary hemostasis in bleeding patients. We tested whether this strategy reverses the effect of clopidogrel using a parallel plate flow chamber model. METHODS: Whole blood from patients, who received a loading dose of clopidogrel with 600 mg and an ongoing dual antiplatelet therapy with 75 mg/d clopidogrel and 100 mg/d acetyl salicylic acid, compared with blood from healthy volunteers was examined in a collagen-coated parallel plate flow chamber. Blood was perfused by suction at a shear rate of 300/s, which is equivalent to 14 dynes/cm to resemble shear stress in conduit arteries. Platelet-covered area, individual thrombus size, and the average thrombus size were assessed morphometrically. The equivalent of 2 or 5 units of PC and/or 2 U/mL of vWF concentrate were used in an attempt to restore coagulation capacity in blood samples of clopidogrel-treated patients. RESULTS: In this model, clopidogrel reduced the increase of thrombus size. The equivalent of 2 U of PC or 2 U/mL of vWF alone did not show any significant changes in thrombus size. 5 U of PC increased thrombus size in clopidogrel-treated patients (P < .05). Thrombus size in clopidogrel blood was increased by combined PC and vWF treatment (by 50%, P < .05), but this increase did not reach control levels (P < .05). CONCLUSIONS: This flow chamber model is suitable for detection of the antiplatelet effect of clopidogrel. Ex vivo addition of PC or vWF does not overcome the effects of clopidogrel in this model, but the combination of both shows a mild and significant improvement in thrombus size.

A dynamic flow-chamber-based adhesion assay to assess canine platelet-matrix interactions in vitro.

BACKGROUND: Dynamic adhesion assays allow the examination of platelet dysfunction and drug effects on platelet function. OBJECTIVE: The purpose of the study was to optimize several parameters such as type and concentration of collagen, wall shear stress, and the concentration of the platelet-activating agonist in a new biochip perfusion chamber for the study of canine platelets. METHODS: After fluorescent staining of platelets, citrated blood of 10 healthy dogs was perfused through the flow chamber coated with different concentrations of canine or bovine skin collagen. Wall shear stress ranged from 14 to 60 dynes/cm(2). Protease-activating receptor 4 (PAR 4) agonist was used for platelet activation. After perfusion, platelet attachment to the collagen matrix was quantified based on fluorescent imaging. Total platelet covered area and average size of platelet covered areas were measured by planimetry. RESULTS: Canine platelet adhesion was supported by ≥ 200 µg/mL canine collagen, but not bovine skin collagen. Consistent results were obtained with a wall shear stress of 14 dynes/cm(2), whereas higher wall shear stress resulted in increased variability. Platelet activation with PAR 4 agonist increased the total platelet covered area and the average size of platelet covered areas. CONCLUSIONS: This study indicates the need to carefully select collagen type and concentration to assess canine thrombus formation in a dynamic flow chamber. The established method should be a useful tool to determine changes in platelet-matrix interactions as an indicator of platelet activation or platelet dysfunction in dogs.