Are Microparticle Levels Useful for Detecting Thrombosis in Patients with Ventricular Assist Devices?

Introduction:  Ventricular assist devices (VADs) are increasingly being used in the clinical management of patients with heart failure. While effective, there are risks associated with the use of a VAD. One of the most serious risks is pump thrombosis characterized by an organized fibrin deposition in the pump, which requires clinical intervention and/or surgical replacement of the device. Due to the growing number of cardiac patients with an implanted VAD, it is important to understand the susceptibility to hemostatic dysregulation among these patients to prevent complications. In current clinical practice VAD pump thrombosis is detected through an elevation in plasma lactate dehydrogenase levels [LDH; >2x upper limit of normal (ULN)]. However, this method of detection is limited by an inability to detect a pump thrombus before it becomes clinically significant. Cellular microparticles (MPs) are submicron membrane-derived exocytotic vesicles that are derived from a variety of cell types by outward blebbing of the plasma membrane in response to activation stimuli. Blood cell-derived MPs can form in response to hemostatic activation, inflammation and altered rheology. The aim of this study was to determine whether levels of cellular MPs can be used more effectively than clinical parameters to predict thrombotic events in VAD patients.

Methods:  Blood samples were collected peri-operatively and long-term post-operatively (until transplant or expiration) during normal clinic visits from 13 consented patients who were implanted with a HeartMate II LVAD (Thoratec, Pleasanton, CA). Fresh whole blood collected in 3.2% sodium citrate was centrifuged for platelet poor plasma then ultracentrifuged (20,000 g, 90 min) to pellet MPs. Aliquots of the MP rich samples were stained with the dye PKH67 (to identify biological membranes) then separated into five tubes containing either: Tyrode’s buffer (control), CD41-PE (platelets), CD45-PE [leukocytes (WBC]), CD146-PE [endothelium (EC)], or CD235-PE [erythrocytes (RBCs)]. Samples were analyzed on an EPICS XL flow cytometer (Beckman-Coulter, Miami, FL) to determine the presence and quantity of cellular MPs using an in-house assay. MPs were identified as PKH67(+) events with a size < 1µm based on size-defined polystyrene beads. Blood samples from 12 healthy individuals were processed in the same manner to establish normal MP levels. A database of clinical events in the LVAD patients was created from medical chart review and included LDH, platelet count, aPTT, D-dimer, fibrinogen and assessment of warfarin by INR. For each patient, clinical and MP data were graphed on a linear time scale; the day that the MP and the clinical values exceeded their ULN was identified and related to time of a clinical adverse event.

Results:  MP levels in patients without adverse events during the follow-up period (2/13) remained below the ULN of 500 microparticles. Patients with thrombotic episodes (11/13) had elevated levels (2000 to 9000 microparticles) a median 50 days (range 35-391 days) prior to LDH elevation; MP levels reverted to normal following resolution of the event. Platelet and RBC MPs were elevated earlier and to a greater extent than MPs from WBCs and ECs. Some patients (2/11) only had an elevation of RBC MPs. Platelet counts were elevated up to 50 days post-surgery (12/13 patients), but levels were normal at time of the thrombotic event. There was no obvious association with adverse events for INR time out of therapeutic range or any other clinical parameter.

Discussion:  Microparticle levels in VAD patients with thrombotic events were shown to cross the normal threshold weeks prior to the time when common clinical parameters indicated an abnormality, suggesting that changes in microparticle levels may be useful in predicting thrombotic events. Hypercoagulability in VAD patients can be influenced by a number of factors including exposure of blood to foreign surfaces, altered shear stresses, inflammation and infection. Data from this investigation warrants further study for the incorporation of microparticles into the clinical management of patients with implanted VADs and to determine whether the pattern of change in microparticle subtypes can identify the stimulus for pump thrombosis, leading to more effective prophylactic measures.