Currently used anticoagulants prevent thrombosis but increase bleeding. We show an anticoagulation therapy without bleeding risk based on a plasma protease factor XII function-neutralizing antibody. We screened for antibodies against activated factor XII (FXIIa) using phage display and demonstrated that recombinant fully human antibody 3F7 binds into the FXIIa enzymatic pocket. 3F7 interfered with FXIIa-mediated coagulation, abolished thrombus formation under flow, and blocked experimental thrombosis in mice and rabbits. We adapted an extracorporeal membrane oxygenation (ECMO) cardiopulmonary bypass system used for infant therapy to analyze clinical applicability of 3F7 in rabbits. 3F7 provided thromboprotection as efficiently as heparin, and both drugs prevented fibrin deposition and thrombosis within the extracorporeal circuit. Unlike heparin, 3F7 treatment did not impair the hemostatic capacity and did not increase bleeding from wounds. These data establish that targeting of FXIIa is a safe mode of thromboprotection in bypass systems, and provide a clinically relevant anticoagulation strategy that is not complicated by excess bleeding.

Background Exaggerated and prolonged inflammation after myocardial infarction (MI) accelerates left ventricular remodeling. Inflammatory pathways may present a therapeutic target to prevent post-MI heart failure. However, the appropriate magnitude and timing of interventions are largely unknown, in part because noninvasive monitoring tools are lacking. We here employed nanoparticle-facilitated silencing of CCR2, the chemokine receptor that governs inflammatory Ly-6Chigh monocyte subset traffic, to reduce infarct inflammation in apoE−/− mice after MI. We used dual target PET/MRI of transglutaminase factor XIII (FXIII) and myeloperoxidase (MPO) activity to monitor how monocyte subset-targeted RNAi altered infarct inflammation and healing. Methods and Results Flow cytometry, gene expression analysis and histology revealed reduced monocyte numbers and enhanced resolution of inflammation in infarcted hearts of apoE−/− mice that were treated with nanoparticle-encapsulated siRNA. To follow extracellular matrix crosslinking non-invasively, we developed a fluorine-18 labeled PET agent (18F-FXIII). Recruitment of MPO-rich inflammatory leukocytes was imaged using a molecular MRI sensor of MPO activity (MPO-Gd). PET/MRI detected anti-inflammatory effects of intravenous nanoparticle-facilitated siRNA therapy (75% decrease of MPO-Gd signal, p<0.05) while 18F-FXIII PET reflected unimpeded matrix crosslinking in the infarct. Silencing of CCR2 during the first week after MI improved ejection fraction on day 21 after MI from 29 to 35% (p<0.05). Conclusion CCR2 targeted RNAi reduced recruitment of Ly-6Chigh monocytes, attenuated infarct inflammation and curbed post-MI left ventricular remodeling.

Background-Blood coagulation is a tightly regulated process of sequentially activated serine proteases culminating in fibrin formation, which is critical for limiting posttraumatic blood loss but also may contribute to acute thrombotic diseases, most notably myocardial infarction and stroke. Recent studies with factor XII-deficient mice revealed that the factor XII-induced intrinsic coagulation pathway is essential for pathological thrombus formation but dispensable for hemostasis. Consequently, these findings led to the hypothesis that factor XII could be a promising pharmacological target for safe antithrombotic therapy. Methods and Results-The complementary DNA of the previously described factor XIIa inhibitor Infestin-4, cloned from the midgut of Triatoma infestans, was fused to recombinant human albumin (rHA) and analyzed in vitro. The resulting protein rHA-Infestin-4 specifically inhibits factor XIIa and causes prolonged activated partial thromboplastin time in human, mouse, and rat plasma. To assess its inhibitory potency in vivo, mice and rats were injected with rHA-Infestin-4 and challenged in pathological thrombus formation models. In addition, bleeding assays were performed. rHA-Infestin-4 completely abolished occlusive arterial thrombus formation in mice and rats while leaving hemostasis fully intact. Furthermore, rHA-Infestin-4 was highly protective in a murine model of ischemic stroke. Conclusion-These results identify rHA-Infestin-4 as a promising agent to achieve powerful protection from ischemic cardiovascular and cerebrovascular events without affecting hemostasis. (Circulation. 2010;121:1510-1517.)

Background-Identification of key molecular players in myocardial healing could lead to improved therapies, reduction of scar formation, and heart failure after myocardial infarction (MI). We hypothesized that clotting factor XIII (FXIII), a transglutaminase involved in wound healing, may play an important role in MI given prior clinical and mouse model data. Methods and Results-To determine whether a truly causative relationship existed between FXIII activity and myocardial healing, we prospectively studied myocardial repair in FXIII-deficient mice. All FXIII Ϫ/Ϫ and FXIII Ϫ/ϩ (FXIII activity Ͻ5% and 70%) mice died within 5 days after MI from left ventricular rupture. In contradistinction, FXIII Ϫ/Ϫ mice that received 5 days of intravenous FXIII replacement therapy had normal survival rates; however, cardiac MRI demonstrated worse left ventricular remodeling in these reconstituted FXIII Ϫ/Ϫ mice. Using a FXIII-sensitive molecular imaging agent, we found significantly greater FXIII activity in wild-type mice and FXIII Ϫ/Ϫ mice receiving supplemental FXIII than in FXIII Ϫ/Ϫ mice (PϽ0.05). In FXIII Ϫ/Ϫ but not in reconstituted FXIII Ϫ/Ϫ mice, histology revealed diminished neutrophil migration into the MI. Reverse transcriptase-polymerase chain reaction studies suggested that the impaired inflammatory response in FXIII Ϫ/Ϫ mice was independent of intercellular adhesion molecule and lipopolysaccharideinduced CXC chemokine, both important for cell migration. After MI, expression of matrix metalloproteinase-9 was 650% higher and collagen-1 was 53% lower in FXIII Ϫ/Ϫ mice, establishing an imbalance in extracellular matrix turnover and providing a possible mechanism for the observed cardiac rupture in the FXIII Ϫ/Ϫ mice. Conclusions-These data suggest that FXIII has an important role in murine myocardial healing after infarction.