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Blood, Vol. 112, Issue 1, 90-99, July 1, 2008
Identification of a fibrin-independent platelet contractile mechanism regulating primary hemostasis and thrombus growth
Blood Ono et al.
112: 90
Supplemental materials for: Ono et al
Files in this Data Supplement:
- Video 1. Thrombus contraction in native blood perfused over a collagen matrix (WMV, 2.06 MB)
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Native (unanticoagulated) human whole blood was perfused through bovine Type 1 collagen-coated (2 mg/ml) glass microslides at 1800 s−1. Thrombus development and fibrin formation was imaged in real-time using DIC microscopy, on an inverted Leica microscope with 63× (1.2 NA) water objective. Video was captured using a Dage-MTI charge-coupled device (CCD) camera 300 ETRCX (Dage-MTI, Michigan City, IN), and is shown here at 10× original speed. The direction of blood flow is indicated. Note – Platelet thrombus contraction appears to commence relatively early, before apparent fibrin formation.
- Video 2. Thrombus contraction occurs independent of fibrin formation or thrombin-mediated platelet activation (WMV, 5.47 MB)
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Lepirudin (800 U/ml) anticoagulated whole blood derived from either humans (A) or PAR4 deficient mice (B) was perfuzed over bovine Type I collagen-coated glass microslides (2 mg/ml) at 1800 s−1. Development and consolidation of thrombi was observed in real-time using DIC microscopy, on an inverted Leica microscope with 63× (1.2 NA) water objective. Video was captured using a Dage-MTI charge-coupled device (CCD) camera 300 ETRCX (Dage-MTI, Michigan City, IN), and is shown here at 10× original speed. The direction of flow is indicated. Note – A representative thrombus is marqueed in the early stages of development, undergoing visible contraction and consolidation.
- Video 3. Rho-kinase promotes the contraction and consolidation of thrombi under flow (WMV, 3.75 MB)
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Lepirudin (800 U/ml) anticoagulated human whole blood was preincubated with either vehicle control (DMSO) or H-1152 (40 µM) for 10min at 37°C, prior to perfusion through bovine Type I collagen-coated glass microslides (2mg/ml) at 1800 s−1. Development and consolidation of thrombi was observed in real-time using DIC microscopy, on an inverted Leica microscope with 63× (1.2 NA) water objective. Video was captured using a Dage-MTI charge-coupled device (CCD) camera 300 ETRCX (Dage-MTI, Michigan City, IN), and is shown here at 10× original speed. The direction of flow in indicated. Note – While the rate of thrombus formation in H1152-treated blood is reduced, thrombi of significant size are able to form. These thrombi are however unstable and unable to form tightly packed platelet aggregates.
- Video 4. The myosin II inhibitor blebbistatin prevents the formation of tightly packed stable thrombi under flow (WMV, 3.77 MB)
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Lepirudin (800 U/ml) anticoagulated human whole blood was preincubated with the myosin II inhibitor, Blebbistatin (200µM) for 10 min at 37°C, prior to perfusion through bovine Type I collagen-coated glass microslides (2mg/ml) at 1800 s−1. Development and consolidation of thrombi was observed in real-time using DIC microscopy, on an inverted Leica microscope with 63× (1.2 NA) water objective. Video was captured using a Dage-MTI charge-coupled device (CCD) camera 300 ETRCX (Dage-MTI, Michigan City, IN), and is shown here at 10× original speed. The direction of flow in indicated. Note – Significant platelet accrual is observed in Blebbistatin-treated whole blood, however, thrombi do not consolidate, and remain loosely packed. As a result, thrombi are highly unstable with continuous embolization of platelet aggregates.
- Video 5. Thrombus contraction occurs in vivo (WMV, 2.17 MB)
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C57BL6 (A) or PAR4-deficient mice (B) (15∼18g) were anaesthetized using 60 mg/kg sodium pentobarbitone, and mesenteric vessels exteriorized and maintained as outlined under ‘Materials and Methods’. A microinjection needle attached to a micromanipulator was used to introduce a puncture injury to a selected mesenteric venule (∼100 µm in diameter) and subsequent thrombus formation and contraction observed in real-time using an inverted Leica microscope with 63× (1.2 NA) water objective. Video was captured using a Dage-MTI charge-coupled device (CCD) camera 300 ETRCX (Dage-MTI, Michigan City, IN), and is shown here at 5× original speed. The direction of flow is indicated. Note - the original thrombus is outlined in white/arrows and undergoes substantial contraction.
- Video 6. Inhibition of Rho-kinase rapidly destabilizes thrombi in vivo (WMV, 5.68 MB)
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A C57BL6 mouse (15∼18g) was anaesthetized using 60 mg/kg sodium pentobarbitone, and mesenteric vessels exteriorized and maintained as outlined under ‘Materials and Methods.’ A microinjection needle (position indicated) attached to a micromanipulator was used to introduce a puncture injury to a selected mesenteric venule (∼100µm in diameter) and subsequent thrombus formation and consolidation was observed in real-time. Once formed, stable thrombi were injected with either vehicle control (DMSO) or the Rho-kinase inhibitor H-1152 (0.87mg/kg) (Release of DMSO/H1152). Thrombi were imaged in real-time using an inverted Leica microscope with 63× (1.2 NA) water objective. Video was captured using a Dage-MTI charge-coupled device (CCD) camera 300 ETRCX (Dage-MTI, Michigan City, IN), and is shown here at 5× original speed. The direction of flow in indicated. Note, while the thrombus exposed to DMSO is stable, the thrombus exposed to the Rho kinase inhibitor (H1152) embolizes rapidly. Repetitive cycles of thrombus growth and embolization were achieved with regular cycles of H1152 administration.
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