Targeting recombinant thrombomodulin fusion protein to red blood cells provides multifaceted thromboprophylaxis.

Thrombin generates fibrin and activates platelets and endothelium, causing thrombosis and inflammation. Endothelial thrombomodulin (TM) changes thrombin's substrate specificity toward cleavage of plasma protein C into activated protein C (APC), which opposes its thrombotic and inflammatory activities. Endogenous TM activity is suppressed in pathologic conditions, and antithrombotic interventions involving soluble TM are limited by rapid blood clearance. To overcome this problem, we fused TM with a single chain fragment (scFv) of an antibody targeted to red blood cells. scFv/TM catalyzes thrombin-mediated generation of activated protein C and binds to circulating RBCs without apparent damage, thereby prolonging its circulation time and bioavailability orders of magnitude compared with soluble TM. In animal models, a single dose of scFv/TM, but not soluble TM, prevents platelet activation and vascular occlusion by clots. Thus, scFv/TM serves as a prodrug and provides thromboprophylaxis at low doses (0.15 mg/kg) via multifaceted mechanisms inhibiting platelets and coagulation.

Analysis of the spatial and temporal characteristics of platelet-delivered factor VIII-based clots.

Normally factor (F) VIII is not expressed in megakaryocytes, but when human FVIII was transgenically expressed in murine megakaryocytes, it was stored in platelet alpha-granules and released at sites of injury. This platelet FVIII (pFVIII) is effective in correcting hemostasis, even in the presence of circulating inhibitors, so it offers a potential gene therapy strategy for hemophilia A. To understand clot development by pFVIII, we have examined clot response to laser injury in both cremaster arterioles and venules in FVIII(null) mice either infused with FVIII or transgenic for pFVIII. In both sets of vessels, pFVIII is at least as effective as infused FVIII. However, there are temporal and spatial differences in fibrin and platelet accumulation within clots depending on how FVIII is delivered. These differences may be related to the temporal and spatial distribution of the alpha-granular-released FVIII within the developing clot, and may explain the increased frequency and size of embolic events seen with pFVIII. These observations may not only have implications for the use of pFVIII in gene therapy for hemophilia A, but may also have physiologic consequences, explaining why many procoagulant factors are delivered both in the plasma and in platelet alpha-granules.