Imaging of Coagulation Reactions During Thrombus Formation In Vivo with Novel Fluorescent Protein Derivatives

Abstract 817

Significant gaps remain in the understanding of how blood cells and the vasculature differentially support coagulation enzyme complex function leading to regulated thrombus formation in vivo. These gaps partly result from the lack of analytic tools with appropriate sensitivity for incisive conclusions. Here we have combined state of the art confocal fluorescent microscopy, established mouse models, and unique recombinant clotting factors as direct imaging probes to advance our understanding of this process. Hemophilic mice typically do not form stable thrombi following microscopic laser injury in the cremaster model. However, thrombus formation in either hemophilia A (HA) or hemophilia B (HB) mice can be restored by infusion of factor Va. Such rescue implies the assembly of prothrombinase and the formation of sufficient thrombin to permit thrombus formation. We employed this injury model using HB mice as a platform to assess the localization of site-specific fluorescent derivatives of Va and Xa in the growing thrombus. We have prepared a FV variant (Va-810_SYA) that is constitutively Va-like with three free cysteines mutated while leaving a single free cysteine at position 539. This site was labeled with Alexa₄₈₈-maleimide without affecting cofactor function. We also employed Xa bearing Alexa₄₈₈ or Alexa₆₄₇ tethered to the active site with a peptidyl chloromethyl ketone. The resulting Alexa₄₈₈-EGR-Xa or Alexa₆₄₇-EGR-Xa derivatives are inactive but retain the ability to assemble into prothrombinase. Co-infusion of Alexa₅₅₅-labeled anti-CD41 antibody to visualize platelets and Alexa₄₈₈-Va-810_SYA (15 μg; n = 4 mice; 20 injuries) produced a robust signal indicating the accumulation of Va-810_SYA and platelets at the site of microscopic laser injury. Similarly, infusion of Va-810_SYA (15 μg) with Alexa₄₈₈-EGR-Xa (2 μg) into HB mice (n = 3; 19 injuries) revealed a strong signal denoting the ability to directly image Xa at the site of vascular damage. Because thrombus formation in these animals requires the infusion of Va and the assembly of prothrombinase, at least part of the Va and Xa visualized in the vicinity of the thrombus must be functional rather than adventitiously bound. Similar results were obtained in wild type mice (n = 3; 21 injuries) using Alexa₄₈₈-Va-810_SYA or Alexa₆₄₇-EGR-Xa indicating that these findings are not peculiar to HB animals. More detailed measurements were pursued by three-dimensional (3-D) confocal imaging analysis 4 min post-injury with fluorescent derivatives of Va, Xa, platelets or fibrin. As expected, platelets and fibrin were limited to the site of microscopic injury. Factors Va and Xa were also found with platelets and fibrin; however these proteins surprisingly were also distributed away from the thrombus on vascular surfaces. These results suggest that surfaces other than platelets, such as the endothelium, can accommodate the assembly and function of Va and Xa. To test this, HB mice (n=3; 15 injuries) were infused with Va or Xa along with integrilin to inhibit platelet function. Consistent with prior results in PAR4^−/− mice, the reduction of platelets at the site of microscopic injury with integrilin had no obvious effect on fibrin deposition. Despite this reduction in platelets, the accumulation of Va or Xa bound in the vicinity of the injury site was not significantly altered providing an explanation for the unchanged levels of fibrin. These unexpected results question the predominant role ascribed to activated platelets in supporting the assembly and function of procoagulant enzyme complexes. Overall these studies indicate that site-specific fluorescent derivatives of Va and Xa can be visualized with appropriate sensitivity and represent powerful tools to establish their spatial distribution at the site of laser injury in the mouse cremaster model. Our approach provides new biological insights into the integrated behavior of the system of enzymic reactions and their modulation by blood cells which lead to thrombus formation in vivo.