Prothrombotic activity of cytokine-activated endothelial cells and shear-activated platelets in the setting of ventricular assist device support

https://doi.org/10.1016/j.healun.2019.02.009Get rights and content

BACKGROUND

We systematically analyzed the synergistic effect of: (i) cytokine-mediated inflammatory activation of endothelial cells (ECs) with and (ii) shear-mediated platelet activation (SMPA) as a potential contributory mechanism to intraventricular thrombus formation in the setting of left ventricular assist device (LVAD) support.

METHODS

Intact and shear-activated human platelets were exposed to non-activated and cytokine-activated ECs. To modulate the level of LVAD-related shear activation, platelets were exposed to shear stress patterns of varying magnitude (30, 50, and 70 dynes/cm2, 10 minutes) via a hemodynamic shearing device. ECs were activated via exposure to inflammatory tumor necrosis factor-α (TNF-α 10 and 100 ng/ml, 24 hours), consistent with inflammatory activation recorded in patients on LVAD circulatory support.

RESULTS

Adhesivity of shear-activated platelets to ECs was significantly higher than that of intact/unactivated platelets, regardless of the initial activation level (70 dynes/cm2 shear-activated platelets vs intact platelets: +80%, p < 0.001). Importantly, inflammatory activation of ECs amplified platelet prothrombinase activity progressively with increasing shear stress magnitude and TNF-α concentration: thrombin generation of 70 dynes/cm2 shear-activated platelets was 2.6-fold higher after exposure and adhesion to 100 ng/ml TNF-α‒activated ECs (p < 0.0001).

CONCLUSIONS

We demonstrated synergistic effect of SMPA and cytokine-mediated EC inflammatory activation to enhance EC‒platelet adhesion and platelet prothrombotic function. These mechanisms may contribute to intraventricular thrombosis in the setting of mechanical circulatory support.

Section snippets

Methods

To evaluate EC‒platelet prothrombotic interaction mechanisms, the following 3 experimental protocols were performed: Protocol 1: The effect of differing extents of SMPA induced by platelet exposure to 30-, 50-, and 70-dynes/cm2 time-constant shear stress patterns for 10 minutes in a hemodynamic shearing device42 was examined; specifically, we evaluated adhesion of shear-activated platelets to untreated ECs. Platelet prothrombinase activity was also evaluated via the platelet activity state

Shear-mediated platelet activation

The level of SMPA increased progressively with increasing shear stress magnitude. Specifically, PAS values of platelets stimulated in the hemodynamic shearing device (HSD) with different levels of shear stress (30, 50, and 70 dynes/cm2) were significantly higher than basal values measured in control/non-stimulated platelet samples (p < 0.0001; Figure 1A). PAS values of platelets stimulated at 30 and 50 dynes/cm2 were comparable (p > 0.05; Figure 1A), whereas thrombin generation of platelets

Discussion

In this study we have examined the potential synergistic effects of mechanical (shear-mediated) platelet activation (SMPA) and inflammatory (cytokine-mediated) EC activation as to their functional interaction to enhance EC‒platelet surface adhesion and prothrombosis.

We observed that shear-activated platelets bind to ECs regardless of the levels of shear activation and EC activation; that is, they bind to non-activated ECs as well. As such, ECs are a pro-adhesive substrate for shear-activated

Study limitations

In this study human umbilical vein endothelial cells (HUVECs) were utilized as a model for endocardial ECs to examine the effect of cytokine activation. Despite differences between the 2 cell types, HUVECs are a general but reliable model to the study of TNF-α‒mediated activation of endocardial ECs. Indeed, HUVECs have been shown to be responsive to physiologic and/or pathologic stimuli and to express those endothelial markers and signaling molecules we sought to analyze, namely ICAM-1, VCAM-1,

Conclusions

We have provided new mechanistic insights into the prothrombotic interaction of mechanical—shear-mediated—stimulation of platelets with inflammatory activation of ECs. Our findings demonstrate a synergy of this interaction in enhancing overall prothrombosis. Specifically, our results provide evidence that EC activation—cytokine-mediated—combines with shear-mediated platelet activation to drive and sustain platelet adhesion to the inflamed endothelium and enhance prothrombotic activity. We have

Disclosure statement

The authors have no conflicts of interest to disclose. This study was supported by Fondazione CARIPLO (2015-1044 to F.C. and M.J.S.), Fondazione Cariplo and Regione Lombardia (2016-0901 to A.R.), the National Institutes of Health (NIH Cardiovascular Biomedical Engineering Training Grant T32 HL007955 to K.R.A.), the National Institute of Biomedical Imaging and Bioengineering (Quantum Grant Award 5U01EB012487-00 to D.B. and M.J.S.), and the Arizona Center for Accelerated Biomedical

Supplementary data

Supplementary data associated with this article can be found in the online version at www.jhltonline.org/.

References (62)

  • S Taghavi et al.

    Surgical technique influences HeartMate II left ventricular assist device thrombosis

    Ann Thorac Surg

    (2013)
  • N Uriel et al.

    Device thrombosis in HeartMate II continuous-flow left ventricular assist devices: a multifactorial phenomenon

    J Heart Lung Transplant

    (2014)
  • AH Bruggink et al.

    TNFalpha in patients with end-stage heart failure on medical therapy or supported by a left ventricular assist device

    Transpl Immunol

    (2008)
  • V Modur et al.

    Endothelial cell inflammatory responses to tumor necrosis factor alpha. Ceramide-dependent and -independent mitogen-activated protein kinase cascades

    J Biol Chem

    (1996)
  • D Bluestein et al.

    Device thrombogenicity emulation: a novel methodology for optimizing the thromboresistance of cardiovascular devices

    J Biomech

    (2013)
  • JJ Eulert-Grehn et al.

    A case of an obstructive inflow thrombus in a HeartMate 3 from the left ventricle into the pump

    J Heart Lung Transplant

    (2018)
  • JM Stulak et al.

    Treatment of device thrombus in the HeartWare HVAD: success and outcomes depend significantly on the initial treatment strategy

    J Heart Lung Transplant

    (2015)
  • R John et al.

    Activation of endothelial and coagulation systems in left ventricular assist device recipients

    Ann Thorac Surg

    (2009)
  • I Netuka et al.

    Outcomes in HeartMate II patients with no antiplatelet therapy: 2-year results from the European TRACE Study

    Ann Thorac Surg

    (2017)
  • L Valerio et al.

    Aspirin has limited ability to modulate shear-mediated platelet activation associated with elevated shear stress of ventricular assist devices

    Thromb Res

    (2016)
  • L Valerio et al.

    Routine clinical anti-platelet agents have limited efficacy in modulating hypershear-mediated platelet activation associated with mechanical circulatory support

    Thromb Res

    (2018)
  • V Modur et al.

    Endothelial cell inflammatory responses to tumor necrosis factor alpha. Ceramide-dependent and -independent mitogen-activated protein kinase cascades

    J Biol Chem

    (1996)
  • RC Starling et al.

    Unexpected abrupt increase in left ventricular assist device thrombosis

    N Engl J Med

    (2014)
  • V Tarzia et al.

    Hemorrhage and thrombosis with different LVAD technologies: a matter of flow?

    Ann Cardiothorac Surg

    (2014)
  • AC Lopilato et al.

    Incidence and risk factor analysis for gastrointestinal bleeding and pump thrombosis in left ventricular assist device recipients

    Artif Organs

    (2015)
  • ME Nassif et al.

    Relationship between anticoagulation intensity and thrombotic or bleeding outcomes among outpatients with continuous-flow left ventricular assist devices

    Circ Heart Fail

    (2016)
  • LT Yarboro et al.

    Pre-implant left ventricular apex position predicts risk of HeartMate II pump thrombosis

    J Card Surg

    (2017)
  • JJ Han et al.

    Higher body mass index increases risk of heartmate ii pump thrombosis but does not adversely affect long-term survival

    Circ J

    (2017)
  • PM Eckman et al.

    Bleeding and thrombosis in patients with continuous-flow ventricular assist devices

    Circulation

    (2012)
  • Selmi M, Chiu WC, Chivukula, VK, et al. Blood damage in left ventricular assist devices (LVADs): pump thrombosis or...
  • J Sheriff et al.

    Comparative efficacy of in vitro and in vivo metabolized aspirin in the DeBakey ventricular assist device

    J Thromb Thrombolysis

    (2014)
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