Platelets and Endothelial Cells Act in Concert to Delay Thrombolysis – Evidence from an In Vitro Model of the Human Occlusive Thrombus^[*]

Summary

The molecular and cellular mechanisms that over a period of hours render a human thrombus progressively resistant to fibrinolysis have been probed with a novel in vitro model. The kinetics of clot formation and fibrinolysis were monitored by laser light scattering with platelet-rich model thrombi contained in cylindrical flow chambers. In selected experiments, human umbilical vein endothelial cells were also cultured to confluence on the inner walls of these “glass blood vessels”. Following an “aging” period (0.5, 2 or 4 h), each thrombus was gently perfused with a bolus of plasminogen/recombinant tissue plasminogen activator to induce fibrinolysis. Platelets delayed lysis of 2 h-aged thrombi by ~70% and (non-stimulated) endothelial cells by ~30%, compared to cell-free control clots. However, even greater lytic delays (~260%) resulted when both vascular cells were present in the same 2 h-aged thrombus. In contrast, rapid lysis was consistently achieved with R298E,R299E t-PA, a genetically engineered plasminogen activator that is insensitive to inhibition by plasminogen activator inhibitor type 1. These observations suggest platelets and endothelial cells act in concert to enrich the fibrin scaffold of an aging human thrombus in plasminogen activator inhibitor. We propose that the presence of both platelets and endothelial cells may contribute to progressive thrombolytic resistance.

^* Supported by Grant # 90-IN-5 from the NC Board of Science and Technology, Research Grant # CA12197 from the National Institutes of Health (Bethesda, MD), the Wake Forest University School of Medicine Interim Funding Program, NATO Grant for International Collaboration in Research # CRG 950213, and Grant NC-96-GS-72 from the American Heart Association, North Carolina Affiliate.

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