Page 261 - Haematologica - Vol. 105 n. 6 - June 2020

APC-I73N variant
confirm the previous hypothesis that a co-factor function for protein S is to enhance the affinity of APC for nega- tively charged membranes. It was interesting to note that the defect in the anticoagulant function of APC-I73N toward FVa in the presence of excess protein S was not
A
recovered, possibly suggesting that co-factor-mediated topographical changes in the active-site pocket that is required for optimal recognition of FVa scissile bond(s) by APC has also been adversely affected for mutant.
Activated protein C sequentially cleaves two peptide
B
Figure 6. Assessment of the activated protein C (APC)-mediated inhibition of thrombin generation. Citrated normal plasma (80 μL) was incubated with 20 μL PPP- Reagent Low (1 pM TF) and APC-WT (red circles), APC-I73N (blue circles) or APC-R352Q (green circles): 5 nM APC in (A) and 10 nM APC in (B) or buffer control (black circles). The kinetics of thrombin generation was monitored by measuring the hydrolysis of a fluorogenic thrombin substrate. (C) The lag time (LT, min), peak height (Peak, nM), time to peak (ttPeak, min) and endogenous thrombin potential (ETP, nM*min) were deduced from thrombin generation curves as described in “Methods”. min: minutes.
AB
C
Figure 7. Assessment of the activated protein C (APC)-mediated inhibition of thrombin generation in protein C-deficient plasma. Citrated protein C-deficient plasma (80 μL) was supplemented with buffer control (black circles), protein C-wild type (WT) (red circles), or protein C-I73N (blue circles) (60 nM each) and either 2 nM sTM (A) or 5 nM sTM (B) and incubated with 20 μL PPP-Reagent Low (1 pM TF). The kinetics of thrombin generation were monitored by measuring the hydrolysis of a flu- orogenic thrombin substrate. (C) The lag time [LT, minutes (min)], peak height (Peak, nM), time to peak (ttPeak, min) and endogenous thrombin potential (ETP, nM*min) were deduced from thrombin generation curves as described in “Methods”. min: minutes.
C
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