Abstract

Although anucleate, platelets are recognized to undergo apoptotic-like events, with some characteristics of activated platelets resembling those of apoptotic nucleated cells, e.g. membrane blebbing (microparticle (MP) formation), exposure of phosphatidylserine (PS). There are reports in the older literature that platelets from BSS patients expose more PS and have enhanced senescence. In the present study, using flow cytometry, we investigated apoptotic-like events, specifically PS exposure, MP formation, cell shrinkage and loss of mitochondrial inner membrane potential (ΔΨm) in platelets from 2 BSS patients (BSS-Ps), compared with platelets from a patient with another inherited macrothrombocytopenia - an MYH9-related disorder (MYH9-RD-P) - and from normal controls. Investigations were done using whole blood to avoid platelet manipulation, especially of the large platelets. Citrated blood was mixed with GPRP and Ca2+, and the agonists collagen (C; 20 μg/mL), thrombin (T; 1 U/mL), SFLLRN (PAR1 activating peptide (AP); 50 μM), AYPGKF (PAR4 AP; 250 μM), or combinations of C with T or the PAR APs. Platelets and MPs were identified as CD41-positive events; platelet size was expressed as median forward scatter (mFSC); MPs were defined as events <0.8 μm; PS exposure was measured as the % platelets with bound fluorescently-labeled annexin A5 and the mean fluorescence intensity (MFI) of bound annexin A5; and loss of ΔΨm was determined by decreased fluorescence of the potential-sensitive probe TMRM concurrently with PS exposure. Resting BSS and MYH9-RD platelets were larger than controls (mFSC of BSS-Ps: 136, 163; MYH9-RD-P: 126; controls: 50±2). Slightly more resting BSS platelets exposed PS than controls, as did MYH9-RD platelets (Table). Activation with C significantly increased the % platelets exposing PS, which was synergistically increased by T or the PAR1 and/or PAR4 APs; more BSS and MYH9-RD platelets exposed PS than did corresponding activated controls (Table).

% PSExposure
Agonist(s)BSS-P1BSS-P2MYH9-RD-Pcontrols
none (resting platelets) 3.0 3.5 3.0 1.3±0.4 
27.0 29.9 37.9 7.0±1.5 
C+T 11.3 35.3 64.5 13.5±1.7 
C+PAR1 AP 20.1 43.6 49.5 16.4±3.1 
C+PAR4 AP 31.6 47.8 49.7 18.4±3.7 
C+PAR1 AP+PAR4 AP 33.4 49.5 54.8 20.6±3.4 
% PSExposure
Agonist(s)BSS-P1BSS-P2MYH9-RD-Pcontrols
none (resting platelets) 3.0 3.5 3.0 1.3±0.4 
27.0 29.9 37.9 7.0±1.5 
C+T 11.3 35.3 64.5 13.5±1.7 
C+PAR1 AP 20.1 43.6 49.5 16.4±3.1 
C+PAR4 AP 31.6 47.8 49.7 18.4±3.7 
C+PAR1 AP+PAR4 AP 33.4 49.5 54.8 20.6±3.4 

Interestingly, the platelets in the PS-exposing subpopulation were small (mFSC of BSS-Ps: 27, 30; MYH9-RD: 32; controls 18±1) and the annexin A5 MFI of this subpopulation of BSS and MYH9-RD platelets was .5-fold higher than that of controls irrespective of the platelet agonist(s). Unstimulated blood samples from patients and controls had 1–2% MPs; adding combinations of agonists led to the highest MP production, with control platelets producing 5–6% MPs, and BSS and MYH9-RD platelets, a 1.5–2.5-fold greater amount. Another apoptosis hallmark, cell shrinkage, was observed upon platelet activation in the non-PS-exposing population; in response to the various agonists, the size of this platelet population in patients and controls correlated inversely with the amount of PSexposing platelets formed (P<0.0.001) and the amount of MPs formed (P<0.0.001). In nucleated cells, dissipation of ΔΨm precedes PS exposure. However, activated BSS, MYH9-RD and control platelets exposed PS with or without a dissipated ΔΨm; ~50% of activated PS-exposing platelets did not exhibit loss of ΔΨm. While only 5–10% of activated non-PS-exposing control or MYH9-RD platelets exhibited ΔΨm loss, a substantial proportion, ~50%, of activated non-PS-exposing BSS platelets had a dissipated ΔΨm; in a rabbit model, we have previously found non-PS-exposing platelets with a collapsed ΔΨm to be associated with platelet senescence. In summary, we show that: PAR1 and PAR4 mediate the synergistic effect of T on C-stimulated PS expression on BSS, MYH9-RD and control platelets; the apoptotic-like event of cell shrinkage occurs in BSS platelets as well as MYH9-RD and control platelets; other such events, PS exposure and MP formation, are enhanced in BSS platelets compared with controls likely due to the large size of BSS platelets since they are also observed in MYH9-RD platelets; activation to expose PS can occur independently of ΔΨm dissipation in BSS, MYH9-RD and control platelets; and the loss of ΔΨm in non-PS-exposing platelets, an apoptotic-like event that is associated with platelet senescence and one that occurs only to a limited extent in MYH9-RD and control platelets, is a previously unrecognized characteristic of BSS platelets.

Disclosures: No relevant conflicts of interest to declare.

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