On this blog we have discussed the rates of activation among hospital blood banks and platelet suppliers, we have given an overview on the sources of platelet activation, and the impact transfusions of activated platelets have on platelet utilization. However, the validity of all these posts hinges on a foundational understanding that microparticle content is a reliable indicator of platelet activation status. For this blog post, we will explore this question: How do we know that microparticle content is a good marker of platelet activation status?

Platelet Activation

The first step in answering this post’s question is to clarify what we mean when we talk about platelet activation. Platelet activation is the natural process during which platelets undergo a drastic change and transition from non-activated, discoid platelets, to an irregular shape with numerous protrusions often called pseudopods (from Greek pseudo “like or fake” and pod “foot”). This shape change is accompanied by numerous other changes including the budding of surface membrane vesicles, the activation of GPIIb/IIIa sites which prime the platelet surface to bind with fibrinogen, and the secretion of many substances from the three types of platelet granules: dense granules, alpha granules, and lysosomal granules.1,2 Platelets are very sensitive, and activation can occur when platelets adhere to exposed collagen or artificial surfaces, shear stress while in circulation, or are exposed to one of a wide range of stimulators including thrombin, ADP, complement, high shear, or low temperature.3-5

The images below show non-activated (left) and activated (right) platelets imaged via transmission electron microscopy (top) and scanning electron microscopy(bottom).

Platelets on right were activated by cold; micrographs provided by E. Maurer-Spurej

Platelet Microparticles

Platelet microparticles (MPs) can be either the result of budded off surface membrane vesicles or intracellular vesicles released during platelet activation. The platelet microparticles that have budded off from the platelet surface maintain a similar surface marker profile as an activated platelet. Platelet MPs have significant pro-coagulant and hemostatic properties3,6,7

In samples of non-activated platelets, very few MPs are present.8,9 Furthermore, 70-90% of MPs in plasma are generated by platelets.10,11 These two findings suggest that the vast majority of circulating MPs are from activated platelets. In addition to MPs formed during activation, in vivo platelet MPs are known to form during platelet storage over 7 days.12 This MP generation during storage is at least partially due to contact with artificial surfaces.13 The additional formation of MP’s during storage is especially prevalent when the platelets are already activated from their donors and are therefore more stress-sensitive.14-17

The special vulnerability of activated platelets to further MP generation helps to explain why we did not find a difference between the activation rates in day 2 platelets versus day 5 platelets. The platelet units that begin storage in a non-activated state typically stay non-activated while the platelet units that begin activated have significant increases in microparticle content but no change in platelet activation status.18

Determining Platelet Activation Status

Due to the very prominent shape change associated with platelet activation, morphological assessment under sufficient magnification and contrast enhancement has served as one of the earliest methods for determining platelet activation status. However, there are significant limitations for the routine use of morphological assessments including the technical difficulty of stabilizing platelets to prevent further activation, and inter-operator variability.

The most common method to determine platelet activation is by flow cytometry. Flow cytometry allows for the determination of platelet activation using labeling with specific antibodies including PAC-1 for detecting the activation of GPIIb/IIIa, CD62P (P-selectin) from alpha granule membranes and CD63 from dense granules.7,19-25 Flow cytometry also gives the ability to determine MP levels of the sample which have been correlated with the expression of many of the above antigens.23-26 There are some limitations to flow cytometry however as it is generally a very technical process that is not adaptable to routine use. Furthermore, the size calibration and gating methods used in flow cytometry mean that often only the largest microparticles are detected 3 Other methods to measure MPs and their association with platelet activation have also been developed,27-30 including dynamic light scattering-based ThromboLUX.14,31-34

Platelet Activation in Diseases

Platelets function both as innate immune cells35-37 and hemostatic cells. Because of this dual role, platelet activation is a defining phenomenon of many pathological conditions. Microparticles are elevated under these pathological conditions25,38-47 which often culminate in thrombocytopenia.48,49 For example, increased platelet activation and elevated platelet-derived microparticle (PMP) levels have been found during acute malaria infection,50 antiphospholipid syndrome,51 gastrointestinal diseases,52-54 psoriasis,55 scleroderma,56 arthritis,57 diabetes,58,59 spontaneous abortion,60 cancer,61,62 thalassemia,63 and cardiovascular pathologies.64-74 Studies have also investigated the diagnostic value of MP changes in response to treatment.75-81

Adenovirus and other viruses induce PMP release during platelet activation82 and promote the formation of platelet-leukocyte aggregates accompanied by cellular microparticle release both in vitro and in vivo.83-85 Increased P-selectin expression by activated platelets in patients with sepsis is associated with increased formation of platelet microparticles, which express surface receptors that enable them to interact with leukocytes.35

Conclusion

Based on published literature, it is well established that platelet activation leads to microparticle generation. The use of MP content as a marker of platelet activation status is supported by the fact that hardly any MPs are present in samples of non-activated platelets and the vast majority of MPs are generated by platelets. The connection between MPs and various disease states further supports the conclusion that MPs are generated during all forms of platelet activation, not just during clotting. The scientific evidence connecting platelet activation and microparticle generation led platelet expert Dr. Alan Michelson to say, “There is no doubt that platelet activation results in microparticles.”

We’d love to hear your input on this subject. If you have any questions or comments about the connection between platelet activation and microparticles please leave a comment in the area below the references.

 

References

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