A new suite of assays are helping identify drug-related effects on hemostasis, which can lead to excessive blood loss or inversely thrombus formation.

Large animal platelet

Pretty much every drug comes with some side-effect. One of the most dangerous side-effects is when the drug alters hemostasis by affecting coagulation factors and/or platelets. Normally, hemostasis keeps blood within damaged vessels after an injury. Few drugs are developed specifically to modify hemostasis, but sometimes a drug can alter hemostasis, leading to unexpected excessive blood loss in patients or the formation of blood clots within the vessels (thrombus). This can limit the drug targets or potentially lead to the withdrawal of the drug from the market.

Because coagulation factors and platelets are the first line of defense against vascular bleeding and are major contributors to thrombosis and inflammation, unexpected effects could lead to risks of thrombus formation or increased bleeding. There are now new, useful methods to assess part of these risks, and we are continuously working on the adaptation of these methods on laboratory species in order to study different types of compounds.

  • Thromboelastography is a method assessing the viscoelastic properties of fibrin clots generated in whole blood samples under conditions of low shear. For this assay, coagulation is initiated in citrated whole blood by addition of calcium within hours after blood draw. The method is a good reflection of wholeblood clotting capacity, as it assesses the contribution of platelets and coagulation factors in the formation of a clot, and provides a dynamic view of clot development and fibrinolysis.
  • Thrombin generation assay measures the overall capacity of the coagulation system to generate thrombin, which is at the center of coagulation pathways and reflects the balance between procoagulant and anticoagulant factors. It provides a unique information that complements the conventional routine coagulation times (prothrombin time (PT) and activated partial thromboplastin time (APTT)), and can reflect the risks of hypercoagulability or hypocoagulability. For this assay, thrombin is generated in fresh or frozen plasma after addition of calcium, phospholipids, and tissue factors, and the kinetics of thrombin formation is measured. Thrombin generation assay is currently under evaluation in clinical trials, and can be evaluated during preclinical studies. This test is a useful tool for the detection of thrombotic events early in the drug development process.
  • Flow cytometric platelet activation evaluates drug-related effects in the first stage of platelet function. Platelet function comprises the following main stages: platelet activation, granules release, and platelet aggregation. When platelets get activated, their cytoplasmic membrane flips, and internal glycoproteins newly exposed on the platelet surface can be visualized by flow cytometry. Flow cytometry is used to evaluate the P-selectin and glycoprotein (GP) IIb-IIIa activation markers, which are trademarks of platelet activation. Platelet activation is quite sensitive and detects the early signals of drug effects on platelets at low concentrations.
  • Platelet aggregometry assesses the later stage of platelet function. When granules are released, platelets start to aggregate. The kinetics of platelet aggregates formation is evaluated on fresh platelet rich plasma or whole blood. This method can be used to detect spontaneous platelet aggregation or decreased platelet aggregation that can be induced by a small molecules or biologics as monoclonal antibodies, or even anti-drug antibodies.

Although these methods have been known for many years in clinics, they are rarely used in preclinical studies. We have improved these assays, and they can be performed during ex vivo and in vivo studies in most laboratory species. It is possible to evaluate ex vivo drug-effects on platelet activation by flow cytometry and platelet aggregation by aggregometry on human blood – on the same day and within the same human or large animal donor. We can also evaluate ex vivo drug effects on coagulation times, thrombin generation, or whole blood clotting on main animal laboratory species. With these methods in place, combinations of drugs may be tested to evaluate their interactive effects on platelets and coagulation factors, and newly developed drugs may be compared to generics. Similarly, we can evaluate the impact of patient plasmas with various sicknesses on healthy donor platelets. All assays may be performed during preclinical in vivo studies in GLP or non GLP settings.

CONCLUSION

Thromboelastography, thrombin generation assay, aggregometry and cytometric detection of platelet reactivity are among the assays that are now used for the characterization of hemostasis, pathologic thrombosis, and refractory hemorrhages. Application of these assays to animal models requires multiple considerations that can be worked through with our teams in order to evaluate the impact of a drug candidate on blood coagulation during in vivo or ex vivo studies.