Development and validation of a high throughput whole blood thrombolysis plate assay
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Key result
A novel high throughput whole blood thrombolysis plate assay was successfully developed, demonstrating that clot maturation significantly delays activation time and reduces maximal clot lysis rate for plasminogen activators.
Population
Citrated whole blood samples from 5 healthy volunteers who had not taken medications affecting haemostasis…
Comparison
In vitro addition of plasmin, urokinase, or… vs Negative control and positive control.
Design
Preclinical
Follow-up
2 hours of spectrophotometric monitoring
Authors
Discussion
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Implication
Enables high-throughput study of clot maturation effects; leaves open clinical translation pending validation.
Structured PICO
P
Population
Blood samples from 5 healthy volunteers were used to develop and validate an in vitro high throughput whole blood thrombolysis plate assay.
I
Intervention
In vitro addition of plasmin (0.01-0.5 U/mL), urokinase (100-400 U/mL), or tissue plasminogen activator (0.35-7 nM) to tissue factor-induced whole blood clots.
C
Comparator
Negative control (PBS without fibrinolytic drug) and positive control (blood without clotting mixture).
O
Outcome
Thrombolysis profile including maximum degradation (Dmax), activation time (At), and maximum clot lysis rate (CLRmax) measured via spectrophotometry.surrogate
The novel high-throughput whole blood halo assay successfully measures detailed thrombolysis kinetics and demonstrates that clot maturation confers resistance to plasminogen activators, which can be partially reversed by GPIIb/IIIa inhibition.
Limitations
- Static conditions of the model do not account for mechanisms involving blood flow
- Need to supplement with calcium chloride and tissue factor with phospholipids generates large amounts of thrombin
- Broad concentration variation of blood components involved in thrombolysis between donors may cause fluctuations
- The halo assay model enables maturation of clots in static conditions and would therefore not be affected by mechanisms involving flow.
- The crosslinking of α2-antiplasmin to fibrin via factor XIII may have no effect in this static model.
Cite This Study
Bonnard et al. (2017) studied Healthy (n=5). Plasmin, urokinase, and tissue plasminogen activator (t-PA) vs. Different doses and clot maturation times was evaluated on Thrombolysis profile (activation time, maximum clot lysis rate, time to 50% lysis). A novel high throughput whole blood thrombolysis plate assay was successfully developed, demonstrating that clot maturation significantly delays activation time and reduces maximal clot lysis rate for plasminogen activators.
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