Two Types of Procoagulant Platelets Are Formed Upon Physiological Activation and Are Controlled by Integrin α _IIb β ₃

Abstract

Objective— Phosphatidylserine (PS) externalization by platelets upon activation is a key event in hemostasis and thrombosis. It is currently believed that strong stimulation of platelets forms 2 subpopulations, only 1 of which expresses PS. Methods and Results— Here, we demonstrate that physiological stimulation leads to the formation of not 1 but 2 types of PS-expressing activated platelets, with dramatically different properties. One subpopulation sustained increased calcium level after activation, whereas another returned to the basal low-calcium state. High-calcium PS-positive platelets had smaller size, high surface density of fibrin(ogen), no active integrin α IIb β 3 , depolarized mitochondrial membranes, gradually lost cytoplasmic membrane integrity, and were poorly aggregated. In contrast, the low-calcium PS-positive platelets had normal size, retained mitochondrial membrane potential and cytoplasmic membrane integrity, and combined retention of fibrin(ogen) with active α IIb β 3 and high proaggregatory function. Formation of low-calcium PS-positive platelets was promoted by platelet concentration increase or shaking and was decreased by integrin α IIb β 3 antagonists, platelet dilution, or in platelets from kindlin-3–deficient and Glanzmann thrombasthenia patients. Conclusion— Identification of a novel PS-expressing platelet subpopulation with low calcium regulated by integrin α IIb β 3 can be important for understanding the mechanisms of PS exposure and thrombus formation.

Procoagulant Platelets Form an α-Granule Protein-covered “Cap” on Their Surface That Promotes Their Attachment to Aggregates

“…Although the procoagulant 'balloon-shaped' platelets have been observed by microscopy for some time (5,8,23), and there have been reports that annexin V is sometimes distributed nonuniformly on their surface (7, 24), we are not aware of any attempts to use confocal microscopy to focus on their 'coating' aspect, whose study has been dominated by flow cytometry (4,(12)(13)(14)(15)17). There was a report on patched fibrinogen distribution on the platelet surface (25); although that phenomenon could be related to the cap described here, the previous study has marked differences: it used weak stimulation with ADP so that there were no coated platelets, there usually were numerous patches instead of single cap, and there was a continuous slow redistribution of fibrinogen in an integrin ␣ IIb ␤ 3 -dependent manner, ultimately resulting in its internalization.…”

“…Although our previous study hypothesized that it can serve to attach coated to non-coated platelets and allow their incorporation into aggregates (8), this hypothesis has not been tested. The vast majority of published results for coated platelets were obtained using flow cytometry (4,6,(12)(13)(14)(15)(16), so the structure and localization of the coat were not studied. One hypothesis for coat formation is that proteins are cross-linked by plateletderived thrombin-activated factor XIII (fXIII) and/or tissue transglutaminase (tTG) (4).…”

Inner Mitochondrial Membrane Disruption Links Apoptotic and Agonist-Initiated Phosphatidylserine Externalization in Platelets

“…16 Unlike apoptotic platelets or platelets stimulated with agonists, PSer externalization in these integrin-dependent procoagulant platelets was associated neither with increased concentrations of cytoplasmic calcium nor with mitochondrial membrane depolarization. Thus, this pathway would represent a novel mechanism of agonist-initiated PSer externalization not associated with a necrotic mechanism of cell death.…”

Procoagulant Platelets

“…Topalov et al 16 observed that high levels of thrombin stimulation produced PS-positive platelets that retained polarized mitochondria and functional GP (glycoprotein) IIb/ IIIa receptors, labeling their new class of platelets as integrinregulated procoagulant platelets. Choo et al observed platelets similar to those of Topalov et al 16 However, closer examination with confocal microscopy and flow cytometry indicated that the experimental conditions used by Topalov et al resulted in microaggregate formation. Aggregates consisted of one procoagulant platelet and at least one nonprocoagulant platelet, thereby, sharing the characteristics of both classes of activated platelets.…”