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Blood, Vol. 111, Issue 7, 3507-3513, April 1, 2008
Determination of surface tissue factor thresholds that trigger coagulation at venous and arterial shear rates: amplification of 100 fM circulating tissue factor requires flow
Blood Okorie et al.
111: 3507
Supplemental materials for: Okorie et al
Files in this Data Supplement:
- Figure S1. Evaluation of TF wash out (JPG, 98.9 KB)
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Whole blood was perfused over TF titration arrays either with a 5-min 1% HSA buffer pre-rinse or with no rinsing. Column averages of fluorescent intensity of the GPIb staining (A) and fibrin(ogen) (B) are plotted against the TF surface concentration on a semi-logarithmic scale for each condition (○ = no rinsing, ● = 5 minute rinse). Data is average ± SEM (N = 30).
- Figure S2. Effect of 100 fM TF on thrombin-antithrombin (TAT) generation or conversion of a fluorogenic thrombin substrate in well plate assay (no flow) (JPG, 174 KB)
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Generation of TAT by 0, 100 fM, or 10 pM of relipidated TF in CTI-treated, recalicified citrated whole blood with time (A). No TAT was detectable over 5 min in the presence or absence of 100 fM TF (A; insert) while 10 pM TF produced abundant TAT within 3 min after addition. Fractional conversion of the fluorogenic substrate boc-VPR-MCA (10 µM) in CTI-treated, recalcified whole blood in the presence or absence of 100 fM TF demonstrated that no thrombin was detectable during the first 5 min (B), while 100 fM TF caused a detectable enhancement of clotting compared to untreated control (C) (n = 4). Method: To detect thrombin activity with time, we added the fluorogenic stubstrate 10 µM boc-VPR-MCA to every well and detected the fluorescence of the released aminomethycoumarin (AMC) with a Thermo Fluoroskan fluorimeter preheated to 37°C every well was read once per minute for 4 hr. After the 4 hours, 5 U thrombin/well was added and then the plate was read again once per min for 20 min to determine the maximal signal to calculate fractional conversion of the peptide substrate.13
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