Calibrated Automated Thrombin Generation in Frozen-Thawed Platelet-Rich Plasma to Detect Hypercoagulability

Abstract

To enhance the practical applicability of the calibrated automated thrombogram (CAT) we investigated whether frozen-thawed platelet-rich plasma (ft-PRP) can be used to assess the function of the protein C inhibitory pathway, while preserving the natural phospholipid composition. Recalcified ft-PRP triggered with 0.5 pM recombinant human tissue factor shows a median thrombin potential of 1,779 nM·min, against 1,576 nM·min for fresh PRP. To obtain ∼70% inhibition, 6.7 nM activated protein C (APC) has to be added, instead of 25 nM in fresh PRP; so the relative APC resistance of PRP appears to depend upon the presence of intact platelets. Factor VIII, added to normal ft-PRP to obtain a concentration of 3.3 U/ml, increases the thrombin potential in the presence of APC 1.5-fold, from 524 to 808 nM·min, in keeping with previously published increases in thrombotic risk in patients with high factor VIII levels. We conclude that thrombography in ft-PRP, with and without added APC, can be used to assess known risk factors for thrombosis, which allows the design of large clinical studies aimed at proving the relationship between thrombin potential and clinical outcome.

Utility of Thrombin-Generation Assay in the Screening of Factor V G1691A (Leiden) and Prothrombin G20210A Mutations and Protein S Deficiency

“…We used the calibrated thrombogram method (5,17 ) to measure thrombin generation continuously in thawed, previously frozen PRP. Briefly, 10 L of buffer (5 g/L bovine serum albumin, 20 mmol/L HEPES, 140 mmol/L NaCl) or 10 L of APC (25 nmol/L; a generous gift from Thomas Lecompte, INSERM U 734, Nancy, France), 10 L of purified recombinant tissue factor diluted in buffer (0.5 pmol/L, a generous gift from Peter Giesen, Maastricht, The Netherlands), and 80 L of thawed PRP were manually pipetted into polypropylene microtiter well plates (Greiner).…”

Reliability of the Thrombin-Generation Assay in Frozen-Thawed Platelet-Rich Plasma

“…Specifically, a thrombin generation lag time Յ1.5 min indicates the need for factor V Leiden genotyping, whereas a peak thrombin concentration Ͼ433 nmol/L indicates the need for factor II G20210A genotyping. As reported in that study, the experiments were performed on thawed, previously frozen, platelet-rich plasma (PRP), and little indication is provided on either the collection procedure or the storage conditions of these samples, both of which are essential requisites to enable reliable TGA results (2,3 ).…”

Thrombomodulin-modified thrombin generation after in vivo recombinant factor VIII treatment in severe hemophilia A

“…10 The replacement of FVIII, both in vitro in FVIII-deficient plasma 11,12 and in vivo in treated hemophilia A patients, 13 is able to restore thrombin generation, while increased FVIII levels may reflect a procoagulant state. [14][15][16] The lag time of the thrombin generation curve was not found to correlate with FVIII levels, and it was suggested recently that slope and time to peak are better predictors of FVIII influences on thrombin generation, 5 tested in hemophilia A patients and healthy controls. Indeed, time to peak was correlated with FVIII levels.…”