Abstract
Background: The generation of thrombin is a critical process in the formation of venous thrombi. In isolated plasma under static conditions, phosphatidylserine (PS)-exposing platelets support coagulation factor activation and thrombin generation; however, their role in supporting coagulation factor binding under shear conditions remains unclear. We sought to determine where activated factor X (FXa), (pro) thrombin, and fibrin(ogen) are localized in thrombi formed under venous shear.
Methodology/Principal Findings: Fluorescence microscopy was used to study the accumulation of platelets, FXa, (pro) thrombin, and fibrin(ogen) in thrombi formed in vitro and in vivo. Co-perfusion of human blood with tissue factor resulted in formation of visible fibrin at low, but not at high shear rate. At low shear, platelets demonstrated increased Ca2+ signaling and PS exposure, and supported binding of FXa and prothrombin. However, once cleaved, (pro) thrombin was observed on fibrin fibers, covering the whole thrombus. In vivo, wild-type mice were injected with fluorescently labeled coagulation factors and venous thrombus formation was monitored in mesenteric veins treated with FeCl3. Thrombi formed in vivo consisted of platelet aggregates, focal spots of platelets binding FXa, and large areas binding (pro) thrombin and fibrin(ogen).
Conclusions/Significance: FXa bound in a punctate manner to thrombi under shear, while thrombin and fibrin(ogen) distributed ubiquitously over platelet-fibrin thrombi. During thrombus formation under venous shear, thrombin may relocate from focal sites of formation (on FXa-binding platelets) to dispersed sites of action (on fibrin fibers).
Methodology/Principal Findings: Fluorescence microscopy was used to study the accumulation of platelets, FXa, (pro) thrombin, and fibrin(ogen) in thrombi formed in vitro and in vivo. Co-perfusion of human blood with tissue factor resulted in formation of visible fibrin at low, but not at high shear rate. At low shear, platelets demonstrated increased Ca2+ signaling and PS exposure, and supported binding of FXa and prothrombin. However, once cleaved, (pro) thrombin was observed on fibrin fibers, covering the whole thrombus. In vivo, wild-type mice were injected with fluorescently labeled coagulation factors and venous thrombus formation was monitored in mesenteric veins treated with FeCl3. Thrombi formed in vivo consisted of platelet aggregates, focal spots of platelets binding FXa, and large areas binding (pro) thrombin and fibrin(ogen).
Conclusions/Significance: FXa bound in a punctate manner to thrombi under shear, while thrombin and fibrin(ogen) distributed ubiquitously over platelet-fibrin thrombi. During thrombus formation under venous shear, thrombin may relocate from focal sites of formation (on FXa-binding platelets) to dispersed sites of action (on fibrin fibers).
| Original language | English |
|---|---|
| Article number | e10415 |
| Number of pages | 8 |
| Journal | PLoS ONE |
| Volume | 5 |
| Issue number | 4 |
| DOIs | |
| Publication status | Published - 29 Apr 2010 |
Bibliographical note
Funding:This work was supported by the American Heart Association (http://www.americanheart.org; M.A.B. - 09PRE2230117 and O.J.T.M. - 09GRNT2150003), the Netherlands Organization for Scientific Research (http://www.nwo.nl; J.W.M.H. and J.M.E.M.C. - 11-400-0076), the Wellcome Trust (http://www.wellcome.ac.uk; J.M.A. and S.P.W. - 073107), and the National Institutes of Health (http://www.nih.gov; P.E.B. - R37HL071544 and RO1HL038779 from the National Heart, Lung, and Blood Institute). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.