Contact activation: important to consider when measuring the contribution of tissue factor-bearing microparticles to
N Boknäs1, L Faxälv, T L Lindahl
1Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden.
Background:
A commercial MP reagent containing phospholipids is used for thrombin generation (TG) measurements to estimate the procoagulant activity of microparticles (MPs). Previous reports have shown that contact activation affects TG when TF levels are low, and that addition of phospholipids might augment this effect.
Objectives:
To quantify the impact of contact activation on TG in the presence of phospholipids and low/no TF, as is the case using a commercially available MP-reagent.
Methods:
Thrombin generation was analyzed using MP- or platelet-rich plasma (PRP)-reagent in the presence and absence of corn trypsin inhibitor and anti-TF antibodies, respectively. To quantify the impact of different experimental parameters on contact activation, microparticle-depleted plasma was analyzed in the presence of different concentrations of phospholipids, TF and/or contact activating agents (kaolin).
Results:
Even with low contact activating blood collection tubes, substantial thrombin generation was observed with the MP-reagent, but this was completely inhibited by addition of corn trypsin inhibitor. Control experiments illustrate that the phospholipids in the reagent play a major role in enhancing TG initiated by FXIIa. Even with the PRP-reagent, which is recommended for determining the content of phospholipids from MPs, TG was partly dependent on contact activation.
Conclusions:
Contact activation plays a major role in TG when using reagents/samples containing phospholipids but little or no tissue factor. This needs to be considered and accounted for in future clinical studies using TG to assess the procoagulant activity of MPs.
Insights
Contact activation significantly impacts thrombin generation (TG) when using phospholipid reagents with low tissue factor (TF). This effect, driven by phospholipids and FXIIa, must be considered in microparticle (MP) studies.
Area of Science:
- Hemostasis and Thrombosis Research
- Biochemistry of Coagulation
- Clinical Diagnostics
Background:
- Commercial microparticle (MP) reagents with phospholipids are used for thrombin generation (TG) to assess MP procoagulant activity.
- Previous studies indicate contact activation influences TG at low tissue factor (TF) levels, potentially augmented by phospholipids.
Purpose of the Study:
- To quantify the effect of contact activation on TG in the presence of phospholipids and minimal or no TF, mimicking conditions with commercial MP reagents.
- Investigate the role of phospholipids and contact activation in TG assays.
Main Methods:
- Thrombin generation (TG) was measured using MP-reagent and platelet-rich plasma (PRP)-reagent.
- Inhibition studies involved corn trypsin inhibitor and anti-TF antibodies.
- Experiments utilized microparticle-depleted plasma with varying concentrations of phospholipids, TF, and contact activators like kaolin.
Main Results:
- Substantial TG was observed with MP-reagent, even with low contact activation, and was fully inhibited by corn trypsin inhibitor.
- Phospholipids in the reagent were identified as key enhancers of TG initiated by FXIIa.
- TG using PRP-reagent also showed partial dependence on contact activation.
Conclusions:
- Contact activation significantly influences TG when using reagents with phospholipids and low/no TF.
- These findings necessitate consideration of contact activation in future clinical studies employing TG to evaluate MP procoagulant activity.
