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.

Perioperative levels and changes of high-sensitivity troponin T are associated with cardiovascular events in vascular surgery patients.

OBJECTIVES: Myocardial infarction after major surgery is frequent, drives outcome, and consumes health resources. Specific prediction and detection of perioperative myocardial infarction is an unmet clinical need. With the widespread use of high-sensitive cardiac troponin T assays, positive tests become frequent, but their diagnostic or prognostic impact is arguable. We, therefore, studied the association of routinely determined pre- and postoperative high-sensitive cardiac troponin T with the occurrence of major adverse cardiac events. DESIGN: This study was a prospective non-interventional trial. SETTING: This study was conducted at Hannover Medical School in Germany. PATIENTS: A total of 455 patients undergoing open vascular surgery were followed for 30 days for the occurrence of major adverse cardiac events. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Preoperative and 24-hour postoperative high-sensitive cardiac troponin T measurements and the respective changes were correlated to medical history and the occurrence of major adverse cardiac events (cardiovascular death, myocardial infarction, and ischemia). Pre- and postoperative high-sensitive cardiac troponin T measurements demonstrated a majority of patients with detectable troponin levels preoperatively and an increase over the 24 hours after surgery. The level of high-sensitive cardiac troponin T was significantly associated with preexisting diseases that constitute the Lee's Revised Cardiac Risk Index. A preoperative high-sensitive cardiac troponin T greater than or equal to 17.8 ng/L and a perioperative high-sensitive cardiac troponin T change greater than or equal to 6.3 ng/L are independently associated with the occurrence of major adverse cardiac events. Adding high-sensitive cardiac troponin T absolute change to the Revised Cardiac Risk Index improves the risk predictive accuracy of the score as evidenced by increased area under receiver operating characteristic and significant reclassification effects. CONCLUSIONS: The risk predictive power of high-sensitive cardiac troponin T change in addition to the Revised Cardiac Risk Index could facilitate 1) detection of patients at highest risk for perioperative myocardial ischemia, 2) evaluation and development of cardioprotective therapeutic strategies, and 3) decisions for admission to and discharge from high-density care units.

Desmopressin (DDAVP) improves recruitment of activated platelets to collagen but simultaneously increases platelet endothelial interactions in vitro.

Platelet dysfunction can cause clinically relevant bleeding. Treatment with DDAVP is advocated for this condition. DDAVP increases von Willebrand factor (VWF) on endothelial cells (ECs) and in plasma. VWF could facilitate platelet deposition on subendothelial collagen. VWF also facilitates platelet/EC interactions. Therefore DDAVP could precipitate thromboembolic events. We used a flow chamber model to study in vitro and ex vivo if DDAVP alters recruitment of platelets to EC and collagen. Resting or TRAP-activated platelets and EC were treated individually or simultaneously with 0.4 ng/ml DDAVP. Fluorophor-labeled platelets (10(6)/ml) were resuspended in reconstituted blood and superfused across EC and collagen in an in vitro flow chamber model at arterial shear (320 s(-1)). Adhesion of platelets to the respective surface was recorded fluorescence microscopically and platelet covered area was assessed. TRAP significantly induced adhesiveness of platelets for collagen and EC. DDAVP pretreatment of platelets did not affect adhesiveness of resting or TRAP-activated platelets for collagen or EC. Adhesiveness of resting but not TRAP-activated platelets was induced on DDAVP-treated EC. DDAVP-conditioned EC supernatant contained vWF and significantly increased platelet deposition on collagen. Platelets from patients with clinically suspected platelet dysfunction undergoing aortic valve replacement exhibited decreased platelet deposition on collagen surfaces. In summary, our data confirm that DDAVP can induce release of platelet adhesion promoting factors from EC, which is most likely vWF. DDAVP has no direct effect on platelets. Blood samples from DDAVP-treated patients do not exhibit significantly augmented platelet deposition on collagen ex vivo. This influence of released promoting factors might cause an increase of undesirable interactions of platelets with EC.

Infection-associated platelet dysfunction of canine platelets detected in a flow chamber model.

BACKGROUND: In the present study, the influence of bacterial infection, lipopolysacharides (LPS) and hydroxyethyl starch (HES) on platelet function in a parallel plate flow chamber were measured. Experiments were performed with non-activated and protease-activating-receptor (PAR) 4 agonist activated platelets. Comparative measurements were in vivo capillary bleeding time, platelet function analyzer and impedance aggregometry. RESULTS: PAR 4 agonist did not increase platelet adhesion of platelets from dogs with bacterial inflammation in the flow chamber in contrast to platelets of healthy dogs. Except from impedance aggregometry with lower sensitivity and specificity, PFA did not detect platelet dysfunctions in dogs with infection. In vitro addition of LPS or HES significantly reduced platelet covered area after PAR-activation. CONCLUSIONS: The flow chamber detects platelet dysfunctions in dogs with inflammatory diseases. In vitro addition of LPS highlights the inhibiting effect of bacterial wall components on platelet function. Platelet dysfunction induced by infection could possibly also be diagnosed after treatment of sepsis with colloids has commenced. The flow chamber could be a useful tool to detect sepsis associated platelet dysfunction given that larger prospective trials confirm these findings from a proof of concept study.

In vivo fluorescence-mediated tomography for quantification of urokinase receptor-dependent leukocyte trafficking in inflammation.

BACKGROUND: Inflammation is characterized by leukocyte recruitment. Macrophages and neutrophils contribute to tissue damage and organ dysfunction. Modulating leukocyte invasion can protect from these adverse effects. Leukocyte recruitment critically depends on the urokinase-type plasminogen activator receptor (u-PAR). We here use a novel technique to longitudinally quantify cell trafficking in inflammatory models in live animals. METHODS: Near-infrared fluorophore-labeled leukocytes were adoptively transferred to mice with thioglycollate peritonitis to study leukocyte trafficking to sites of inflammation. Macrophage and neutrophil trafficking was followed with three-dimensional fluorescence-mediated-tomography. u-PAR-/- and wild-type macrophage recruitment was studied by cross-over adoptive cell transfer to elucidate the role of leukocytic versus u-PAR expressed on other cells. Endotoxic shock-induced pulmonary inflammation was used to study u-PARs role for pulmonary neutrophil recruitment. RESULTS: Mice experiencing peritonitis showed a significant increase in mean fluorescence intensity because of enhanced macrophage (315%, n=9-10), P<0.05) or neutrophil (194%, n=6, P<0.02) recruitment. Fluorescence-mediated-tomography uncovered a macrophage recruitment defect in the peritonitis model for u-PAR-/- mice (147% of baseline) compared with control mice (335% of baseline, n=8-9, P<0.05). When u-PAR-/--macrophages were transferred to wild-type mice fluorescence intensity increased to 145% while wild-type macrophage transfer into u-PAR-/- resulted in 192% increase compared with baseline (n=6, P<0.05). Reduced neutrophil recruitment in pulmonary inflammation in u-PAR-/- mice was accompanied by improved pulmonary gas exchange. CONCLUSION: Using noninvasive in vivo fluorescence-mediated tomography to image leukocyte recruitment in inflammatory mouse models, we describe a novel macrophage recruitment defect in u-PAR-/- mice. Targeting u-PAR for modulation of leukocyte recruitment is a promising therapeutic strategy to ameliorate leukocyte induced tissue damage.

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.