Novel mouse hemostasis model for real-time determination of bleeding time and hemostatic plug composition.

INTRODUCTION: Hemostasis is a rapid response by the body to stop bleeding at sites of vessel injury. Both platelets and fibrin are important for the formation of a hemostatic plug. Mice have been used to uncover the molecular mechanisms that regulate the activation of platelets and coagulation under physiologic conditions. However, measurements of hemostasis in mice are quite variable, and current methods do not quantify platelet adhesion or fibrin formation at the site of injury. METHODS: We describe a novel hemostasis model that uses intravital fluorescence microscopy to quantify platelet adhesion, fibrin formation and time to hemostatic plug formation in real time. Repeated vessel injuries of ~ 50-100 µm in diameter were induced with laser ablation technology in the saphenous vein of mice. RESULTS: Hemostasis in this model was strongly impaired in mice deficient in glycoprotein Ibα or talin-1, which are important regulators of platelet adhesiveness. In contrast, the time to hemostatic plug formation was only minimally affected in mice deficient in the extrinsic tissue factor (TF(low)) or the intrinsic factor IX coagulation pathways, even though platelet adhesion was significantly reduced. A partial reduction in platelet adhesiveness obtained with clopidogrel led to instability within the hemostatic plug, especially when combined with impaired coagulation in TF(low) mice. CONCLUSIONS: In summary, we present a novel, highly sensitive method to quantify hemostatic plug formation in mice. On the basis of its sensitivity to platelet adhesion defects and its real-time imaging capability, we propose this model as an ideal tool with which to study the efficacy and safety of antiplatelet agents.

Effect of p38 MAP kinases on contractility and ischemic injury in intact heart.

The p38 MAP kinases are stress-activated MAP kinases whose induction is often associated with the onset of heart failure. This study investigated the role of p38 MAP kinase isoforms in the regulation of myocardial contractility and ischemia/reperfusion injury using mice with cardiac-specific expression of kinase dead (dominant negative) mutants of p38alpha (p38alphadn) or p38beta (p38betadn). Hearts were subjected to 20 min ischemia and 40 min reperfusion. Immunofluorescence staining for p38alphadn and p38betadn protein was performed on neonatal cardiomyocytes infected with adenovirus expressing flag-tagged p38alphadn and p38betadn protein. Basal contractile function was increased in both p38alphadn and p38betadn hearts compared to WT. Ischemic injury was increased in p38betadn vs. WT hearts, as indicated by lower posti-schemic recoveries of contractile function and ATP. However, despite a similar increase in contractility, ischemic injury was not increased in p38alphadn vs. WT hearts. Immunohistological analysis of cardiomyocytes with comparable levels of protein overexpression show that p38alphadn and p38betadn proteins were co-localized with sarcomeric alpha-actinin, however, p38alphadn was detected in the nucleus while p38betadn was exclusively detected in the cytosol. In summary, attenuated p38 activity led to increased myocardial contractility; specific isoforms of p38 and their sub-cellular localization may have different roles in modulating ischemic injury.

Using a gene-switch transgenic approach to dissect distinct roles of MAP kinases in heart failure.

We have demonstrated that Cre-loxP-mediated gene-switch transgenesis is an effective approach to achieve targeted and temporally regulated gene manipulation in the heart. Using this approach, we have established animal models with targeted activation of different MAPK pathways. From these animal models, we identified distinct features of cardiac pathology associated with individual MAPK branches (summarized in Fig. 8). Specifically, Ras activation appears to promote cardiac hypertrophy, whereas p38 and JNK activation does not. Whereas Ras activation leads to depressed diastolic function associated with suppressed calcium transients and SR calcium uptake, p38 activity seems to modulate cellular contractility without affecting intracellular calcium cycling. Although all three models displayed extensive remodeling in the myocardium, the extent and the composition of interstitial fibrosis are different among them, with Ras- and p38-activated hearts promoting collagen-based fibrosis, and JNK activation leading to induction in fibronectin-based reticular fiber. In addition, JNK activation leads to loss of Cx43 expression and abnormal cell-cell communication. Therefore, ERK, p38, and JNK are three distinct intracellular signaling pathways that contribute to different aspects of cardiac pathology during heart failure. Combining sophisticated genetic manipulation with comprehensive analysis at physiological, molecular, and genomic levels, the transgenic animals established in these studies should serve as valuable model systems to identify and dissect the underlying mechanisms for different aspects of cardiac pathology such as hypertrophy, contractile dysfunction, and abnormal cell-cell communication. The insights learned from these investigations may help to develop novel therapeutic approaches to confront this devastating disease.