Necrotic but Not Apoptotic Platelets Are Functionally Procoagulant
Authors: Kholmukhamedov Andaleb , Jobe Shawn M. 
Published: October 2019

Abstract
There are numerous modes of cell death in mammalian cells. In platelets the primary identified modes are apoptosis and necrosis. Apoptosis is a form of programmed cell death, and in platelets it is considered to be important in their clearance. Besides pyknosis (cell shrinkage) and engulfment by resident phagocytes in vivo, a characteristic feature of apoptosis in nucleated cells is retraction of pseudopodia. However, in platelets, this does not occur. Instead, distinct from that which occurs during activation, lamelopodia and filopodia are not observed in apoptotic platelets suggesting that they have a limited potential to actively participate in coagulation. Necrosis, either regulated or pathologic, is characterized by cytoplasmic swelling (oncosis), swelling of cytoplasmic organelles, and, in later stages, rupture of the plasma membrane. Additionally, secondary necrosis can occur as a consequence of the activation of apoptotic pathways. Apoptosis is an ATP requiring process. In instances when an apoptotic signal was initiated and insufficient ATP is present for the completion of apoptosis, this ATP-depleted cell will undergo death with morphologic features of necrosis. Procoagulant platelets are those platelets that are capable of supporting the assembly of functional tenase and prothrombinase complexes. Due to the increased complexity of experiments demonstrating functional activity, the ability to support procoagulant activity has been presumed in subpopulations of platelets with variable degrees of phosphatidylserine exposure. Here we investigated the procoagulant function and the extent of PSer exposure in platelets undergoing different modes of cell death.
Washed human platelets were stimulated with the BH-3 mimetic ABT-737 (100 nM) (apoptotic initiator) or convulxin (250 ng/mL, CVX) (initiator of regulated necrosis). The extent of PSer externalization was estimated by cytometric evaluation of annexin V (AnnV) binding, and functional activity was assessed using tissue factor-initiated thrombinoscopy. Upon stimulation two distinct subpopulations of PSer exposing platelets with "low" and "high" AnnV binding could be distinguished. In CVX-stimulated platelets only a single subpopulation ("high") of PSer exposing platelets could be distinguished. But in platelets stimulated with the apoptotic initiator ABT-737 both "low" and "high" subpopulations of AnnV binding platelets could readily be discerned. To assess the procoagulant functionality of these various modes of cell death ABT-737 (generating ~38% "high" and ~46% "low" AnnV positive platelets) and CVX (~33% "high" AnnV) stimulated platelets were compared by thrombinoscopy. Relative to unstimulated platelets, inclusion of CVX-stimulated platelets within the reaction significantly shortened both the lag time (~5 to ~3 min (p<0.05)) and time to peak thrombin (~13 to ~8 min (p<0.05)). These shortened response times were accompanied by a ~2-fold increase in peak thrombin (p<0.05). In contrast to the potentiating effects of CVX-stimulation, ABT-737 stimulation had no effect on measures of thrombin; this despite having equivalent (when assessing only high) or even a greater percentage of AnnV binding (when assessing low and high).
These results are the first to differentiate the mode of platelet cell death and its corresponding effects on PSer exposure and a functional measure of procoagulant function. Apoptotic and necrotic platelets are not only distinguished by their molecular mechanism of initiation, but also by their functional ability to support coagulation. Only platelets undergoing cell death by necrotic pattern were functionally procoagulant as measured by direct prothrombinase activity. Advancement in understanding the principles of cell death in procoagulant platelet formation might be beneficial for the determination and guidance of novel pharmacological strategy targeted for the treatment of procoagulant related hematopathologies.

Disclosures
Jobe: CSL: Consultancy; Shire: Consultancy; Octapharma: Consultancy.