Continuous Thrombin Generation in Whole Blood: New Applications

Background.

Hemostatic tests have been utilized to clarify the blood coagulation potential of both healthy and diseased individuals. They include tests for whole blood and plasma clotting times, coagulation and fibrinolytic factors and, recently, thrombin generation (TG). TG assays provide explicit information and remain the most physiologically-relevant hemostatic tests ex vivo. We have currently been using a TG assay in whole blood, described by Ninivaggi et al. (2012), for the evaluation of several hemostatic agents and disorders of blood coagulation.

Methods.

Whole blood from either healthy donors or trauma patients was drawn into 3.2% sodium citrate [± 0.1 mg/mL corn trypsin inhibitor (CTI) prior to the assay; prevents contact pathway activation] or into 3.2% sodium citrate/0.1 mg/mL CTI. Selected agents were added to blood prior to recalcification and TG was monitored in a fluorogenic assay.

Packed red blood cells (pRBC) from healthy donors were reconstituted with platelet-poor plasma (PPP) and treated with 0.1 mg/mL CTI. TG was monitored in the absence of any exogenous initiation, in the presence of 5 pM TF initiation and also in the presence of synthetic phospholipids.

Results.

Whole Blood

A. Blood from 10 healthy donors was collected into both citrate (with CTI added prior to the assay) and CTI/citrate and TG was monitored ± 5 pM relipidated tissue factor (TF) initiation. In the absence of TF, blood collected into citrate led to a lag phase ~3-fold longer than that in the presence of TF and peak thrombin ~1.4-fold lower. Blood collected into CTI/citrate, in the absence of TF, led to a lag phase ~6.4-fold longer than that in the presence of TF and peak thrombin ~4.3-fold lower.

B. Titrations of relipidated TF and FXIa into citrated blood containing CTI led to concentration-dependent changes in the duration of the lag phase.

C. Several TG antagonists were evaluated in citrated blood containing CTI triggered with 5 pM TF.

a. 0.5 U/mL unfractionated heparin prolonged the lag phase ~2-fold and suppressed the peak thrombin ~3-fold.

b. Hemophilia A and B were "induced" via the addition of inhibitory antibodies to FVIII and FIX, respectively, and although the lag phase was not altered both additions led to significant TG suppression with peak thrombin dropping ~3-fold.

c. Several thrombin and FXa inhibitors were evaluated at their pharmacologic concentrations. Fondaparinux slightly prolonged the lag phase, whereas dabigatran increased it by 2.6-fold. Rivaroxaban doubled the lag phase while bivalirudin was slightly less efficient. None of these inhibitors had a pronounced effect on the rate and peak value of TG.

d. Activated protein C (APC) at 10 nM slightly prolonged the lag phase and suppressed the peak thrombin.

D. Citrate blood from a trauma patient was tested for endogenous activity. In the presence of CTI and absence of an initiator, α-TF antibody did not alter TG, indicating the absence of functional TF. α-FXIa antibody prolonged the lag phase and suppressed TG and α-FIXa antibody completely abolished TG, indicating the presence of active FXIa and FIXa.

Packed Red Blood Cells (pRBC)

In the absence of an initiator, no TG was observed in pRBC/CTI reconstituted with PPP. The addition of 5 pM TF led to TG after ~8 min with a peak value slightly lower than in blood (described in A).The addition of phospholipids did not change the TG profile.

Conclusions.

This continuous whole blood TG assay requires 15 µL of blood for a triplicate analysis of a single condition and has the potential for the evaluation of TG in disorders relevant to blood coagulation and for the monitoring of treatments administered in response to these disorders.