Methods of treating thromboembolic disorders
A technique for thromboembolism and thrombus, applied in the field of platelet contractility inhibitor to dissolve thrombus
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Embodiment 1
[0119] Embodiment 1 Materials and methods
[0120] Material
[0121] The Rho kinase inhibitor H1152 was obtained from Toronto Research Chemicals (Canada). IP 3 The receptor antagonist 2-aminoethoxybiphenylborate (2-APB) was from Cayman Chemicals (Michigan, USA), HA1077, the myosin II inhibitor Blebbistatin[-] and its inactive enantiomer Blebbistatin [+] was obtained from Chemicon (USA). DiIC 12 From BD Biosciences (NSW, Australia). Recombinant hirudin (lepirudin) was purchased from Pharmion (Australia).
[0122] mouse strain
[0123] All involved the use of C57Bl6 and PAR4 - / - Procedures for mice were approved by the Alfred Medical Research and Education Precinct (AMREP) Animal Ethics Committee (AEC) (Melbourne, Australia) under project numbers E / 0569 / 2007 / M, E / 0621 / 2007 / M and E / 0464 / 2006 / M.
[0124] Collection of blood, preparation of PRP and washed platelets
[0125] All processes involving the collection of human and mouse blood were supervised by the Monash Un...
Embodiment 2
[0136] Example 2 Identification of the systolic phase during thrombus development
[0137] The importance of platelets in transmitting cytoskeletal contractile forces to fibrin polymers for clot retraction is well established. But the importance of these contractile mechanisms in regulating the various stages of thrombus growth, especially under physiological blood flow conditions, has not been clearly established. To investigate this, the inventors used an in vitro perfusion system that allowed real-time analysis of platelet adhesion and thrombus growth on immobilized type I fibrin collagen substrates. At arteriole shear rate (1800s -1 ) perfusion of native (non-anticoagulated) whole blood causes rapid platelet adhesion and aggregate formation, forming large stable aggregates within 2 minutes of flow. Analysis of deposited fibrin(ogen) by co-infusion of fluorescently-labeled fibrinogen revealed extensive incorporation of fibrin(ogen) within the developing thrombus, with i...
Embodiment 3
[0139] Example 3 The Importance of Rho Kinase to Thrombus Contraction
[0140] Actinomyosin-based contractility is linked to activation of myosin light chain kinase by calcium / calmodulin-dependent activation of myosin light chain kinase and Rho kinase-dependent inactivation of myosin phosphatase Phosphorylation is closely linked. In platelets, calcium activation of myosin light chain kinase appears to be the major contractile mechanism regulating platelet shape change and fibrin clot retraction 4 . To investigate the role of cytosolic calcium influx in modulating thrombus contraction, whole blood perfusion studies were performed to prevent calcium influx (EGTA / MgCl 2 ) or mobilize calcium from internal stores (IP 3 receptor antagonist-2-APB) experimental conditions. Sequestration of extracellular calcium with EGTA significantly reduced thrombus shrinkage (up to 52% at 5 min perfusion, p image 3 A). Under similar experimental conditions, inhibition of calcium mobilizatio...
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