(VP003) A FUNCTIONAL ASSAY TO QUANTIFY THE HEMOSTATIC PROPERTIES OF STIMULATED PLATELETS

Background: Platelets are the smallest anucleate cells in circulation and are a major component of primary hemostasis to minimize blood loss upon vascular damage. They are associated with a number of diseases based on their abnormal function, count, or both. However, low platelet count (thrombocytopenia) does not always translate to bleeding outcomes, suggesting there may be functional differences of the platelets in forming a necessary thrombus. Clinically, functional platelet tests rely on lumi aggregometry or flow cytometry. These tests do not necessarily reflect the hemostatic properties of platelets during coagulation. In addition, how platelets affect not only clot formation but its degradation (fibrinolysis) is often ignored, largely due to complexities associated with the assay design. Furthermore, most tests are unable to probe for functional differences between stimulated and resting platelets since single-stage experiments often require stimulation of all platelets during reaction initiation. Here we report a novel assay design using modified clot lysis assays that can specifically quantify the hemostatic potential of platelets that is conferred upon stimulation.

METHODS AND RESULTS: Washed platelets were obtained from healthy individuals. After letting the isolated platelets rest for at least 30 minutes at room temperature, they were either left untreated or were activated with 30 nM thrombin and incubated for 15 minutes at 25 degrees. After activation, thrombin was inhibited by the addition of excess hirudin. Separately, normal pooled human plasma was prepared for a 1:3 final dilution in the reaction mixture, along with additional hirudin (2.5 µM) and either resting or activated platelets at varying concentrations (ranging between 0 and 1 x 108 platelets/mL). Clots were formed using batroxobin, a thrombin-like enzyme that triggers fibrin formation and polymerization without activating platelets, in the presence of CaCl2 (10 mM). Fibrinolysis was initiated with tPA (0.6 nM). The reactions were monitored using a 96-well plate reader at 1-minute intervals at 400 nm. Lysis times were defined as the time required to reach 50% signal change during clot degradation from the start of the reaction.

Conclusion: In the presence of resting platelets, clot lysis times of batroxobin clots were not affected. However, there was a platelet concentration-dependent lysis time response for activated platelets, particularly sensitive between 0 and 5 x 107 platelets/mL; below what would be considered thrombocytopenic (1.5 x 108 platelets/mL). Therefore, this assay has the potential to quantify platelets based on functional capacity (ie. dysfunctional platelets) and concentration (ie. bleeding risks for patients with thrombocytopenia).

Lay Abstract Content: Purpose of the research:
Platelets are the smallest cells in the blood. While they are involved in many of the body’s responses to infections and distress, they are mostly known for being the key players in blood clotting. Upon injury that leads to blood loss, platelets are the first to respond to the wound. Here, they become activated, which can then form large clusters with other activated platelets to form a plug, in order to stop further blood loss. Once the blood vessel is repaired, other processes are in place to remove unwanted clots in order to restore blood flow. This process is referred to as fibrinolysis. How platelets are directly involved in fibrinolysis is still under investigation. However, we have recently shown that platelets are certainly involved in initiating and up-regulating fibrinolysis under certain conditions. How the balance of blood clotting and fibrinolysis are determined at the platelet-level is unclear.
Clinically, although some tests exist to test for the functionality of platelets, they simply determine their ability to form clusters with each other. Thus, it is not possible to determine functional differences between resting and stimulated platelets. We have developed a novel functional assay that could characterize platelets and their hemostatic potentials. We now aim to further develop this functional assay, which could be a potent tool to identify patients that suffer from blood disorders that stem from dysfunctional platelets.

Relevance of the research:
Platelet disorders that stem from dysfunctional platelets can be challenging to diagnose as counting their numbers alone would not be sufficient. This is also true for disorders such as immune thrombocytopenic purpura (ITP) whereby platelet counts are severely low. However, some patients are prone to bleeding complications while others are not. By specifically probing for the platelets’ ability to promote blood clotting, we may be able to identify those that are at risk for developing blood disorders. Preventatively treating such patients may lead to less down-stream complications and improved quality of life.

General Approach:
Our novel assay relies on being able to differentiate the contributions by resting versus activated platelets. Typically, thrombin is generated by the plasma upon stimulation, which consequently forms clots. However, the same thrombin also globally stimulates platelets, and as such, there has not been a good method to differentiate the functional contributions of platelet to fibrinolysis solely upon activation. Our assay segregates activated versus resting platelets by independently activating the platelets ahead of time with thrombin, and thrombin is inactivated by the addition of a thrombin-specific inhibitor. Resting or pre-activated platelets are then added to plasma, and the plasma clots are formed with batroxobin – a thrombin-like enzyme that can generate a clot but does not affect platelets. Fibrinolysis is then initiated by the presence of a clot-busting drug (tPA). Clot lysis times (CLT) are then determined as the time required to reach half-maximal clot degradation. The standard curve is generated by plotting the CLT as a function of changing platelet counts.