Sergiy Mykhaylov

Thrombin-triggered platelet apoptosis

Summary.  Background: Thrombin is primarily known as a coagulation factor and as an inducer of platelet activation and aggregation. It has been reported that thrombin modulates apoptosis of nucleated cells. Objectives: The current study investigated whether thrombin can affect apoptosis in anucleated human platelets. Methods: Using flow cytometry, we studied platelet apoptosis at the single‐cell level, analyzing markers of mitochondrial and cytoplasmic apoptosis. Western blotting was also employed, in addition to flow cytometry, for determining the expression of Bcl‐2 family proteins. Results: We found that human α‐thrombin induced four key manifestations of apoptosis in human platelets: (i) mitochondrial inner transmembrane potential (ΔΨm) depolarization; (ii) strong expression of pro‐apoptotic Bax and Bak proteins but only weak expression of anti‐apoptotic Bcl‐2 protein; (iii) caspase‐3 activation; and (iv) phosphatidylserine (PS) exposure. Conclusions: This study demonstrates that, aside from its ‘classical’ function as an inducer of platelet activation, thrombin can trigger platelet apoptosis, where it acts as a death ligand. These data indicate that thrombin triggers platelet apoptosis by impacting on several intracellular apoptotic targets, including shifting the balance between Bcl‐2 regulatory proteins in a pro‐apoptotic direction, depolarizing the inner mitochondrial membrane, activating the executioner caspase‐3, and stimulating aberrant exposure of PS on the platelet surface.

Intravenous immunoglobulin inhibits anti-glycoprotein IIb-induced platelet apoptosis in a murine model of immune thrombocytopenia.

We have previously shown that injection of anti‐glycoprotein (GP) IIb induces murine immune thrombocytopenia (ITP) and that intravenous immunoglobulin (IVIg) ameliorates ITP. We hypothesise that murine ITP may be associated with platelet apoptosis, which is upregulated by anti‐GPIIb and downregulated by IVIg. The current study demonstrated that anti‐GPIIb injection induced three critical apoptosis manifestations in platelets: (i) mitochondrial inner transmembrane potential (ΔΨm) depolarisation; (ii) caspase‐3 activation; and (iii) phosphatidylserine (PS) exposure. IVIg administration inhibited caspase‐3 activation and PS exposure, but not ΔΨm‐depolarisation, in anti‐GPIIb‐treated platelets, demonstrating that IVIg ameliorates thrombocytopenia concomitantly with inhibiting late, but not early mechanisms of platelet apoptosis.

Platelet activation and apoptosis are different phenomena: evidence from the sequential dynamics and the magnitude of responses during platelet storage

Guise, T.A. (2006) Bone loss and fracture risk associated with cancer therapy. The Oncologist, 11, 1121–1131. Holick, M.F. (2007) Vitamin D deficiency. New England Journal of Medicine, 357, 266–281. Kyle, R.A., Yee, G.C., Somerfield, M.R., Flynn, P.J., Halabi, S., Jagannath, S., Orlowski, R.Z., Roodman, D.G., Twilde, P. & Anderson, K. (2007) American Society of Clinical Oncology 2007 Clinical Practice Guideline update on the role of bisphosphonates in multiple myeloma. Journal of Clinical Oncology, 25, 2464–2472. Roodman, G.D. (2005) Myeloma bone disease: pathogenesis and treatment. Oncology (Williston Park), 19, 983–984. Terpos, E., Politou, M. & Rahemtulla, A. (2005) The role of markers of bone remodeling in multiple myeloma. Blood Reviews, 19, 125–142.

The GPIIbIIIa antagonist drugs eptifibatide and tirofiban do not induce activation of apoptosis executioner caspase-3 in resting platelets but inhibit caspase-3 activation in platelets stimulated with thrombin or calcium ionophore A23187

Platelet surface receptor, glycoprotein (GP) IIbIIIa (integrin αIIbβ3), mediates platelet aggregation and plays a key role in hemostasis and thrombosis.[1][1],[2][2] Numerous GPIIbIIIa antagonists have been designed and tested as inhibitors of platelet aggregation.[3][3] Two of these antagonists

Stored platelet concentrates produced by the platelet-rich plasma method are more resistant to apoptosis but more sensitive to activation than are platelets prepared by the buffy-coat and apheresis methods.

Recently, we studied the contributions of PLT activation and apoptosis to the PLT storage lesion during conventional (Days 2-5), extended (Days 6-8), and long-term (Days 11-16) storage of leukoreduced PCs produced by the PLT-rich plasma method (PRP-PCs). Four apoptotic parameters, including depolarization of mitochondrial inner membrane potential (DYm), caspase-3 activation, phosphatidylserine (PS) exposure, and release of PLT microparticles, were used for quantifying mitochondrial, cytoplasmic, plasma membrane, and cellular manifestations of PLT apoptosis, respectively, and PLT activation was determined by P-selectin (CD62) exposure. It was demonstrated that PLT activation and apoptosis responses are triggered sequentially, rather than in parallel, during storage of PRP-PCs. PLT activation was readily induced under the conventional and extended PLT storage, whereas triggering of PLT apoptosis required long-term storage. Furthermore, for all storage days, the level of PLT activation reached was significantly higher than the level of apoptosis. Different results have been reported in the subsequent publication of Albanyan and coworkers, in which PLT activation and apoptosis phenomena during storage for 1, 3, 5, and 7 days were studied in leukoreduced PCs produced by the buffy coat (BC-PCs) and apheresis (APPCs) methods. The aim of the current report is to compare PLT activation and apoptosis responses in PRP-PCs versus BC-PCs and AP-PCs by analyzing results of these closely related studies. Table 1 summarizes the reported data on the dynamics of DYm depolarization and caspase-3 activation in PRP-, BC-, and AP-PCs. In PRP-PCs, DYm depolarization and caspase-3 activation did not increase significantly even after long-term storage for up to 12 days; storage for 13-14 days was required for significant triggering of these apoptotic events. In contrast, much faster apoptotic changes were observed during storage of BC-PCs and AP-PCs: when DYm depolarization was measured by the red-to-green fluorescence ratio of the DYmdetecting dye JC-1, as was employed in our study, DYm depolarization was significantly increased by Days 5 and 7 (Table 1). However, when DYm depolarization was measured by an alternative JC-1 method, as the percentage of PLTs with depolarized DYm, which detects cells with full DYm depolarization, only BC-PCs, but not AP-PCs, showed this apoptotic alteration on Days 5 and 7 (Fig. 1B, right panel in Albanyan et al.). As evidenced from the data on the sequential dynamics (Table 1) and the magnitude of PLT apoptosis and activation responses, as measured by PS and CD62 exposure, respectively (Table 2), the ability of PLT apoptosis and activation to be induced by PLT storage differs significantly in PRP-PCs, in comparison to BC-PCs and AP-PCs. In PRP-PCs, PLT apoptosis is more resistant to PLT storage than PLT activation (Table 2, Column 2, p < 0.001 for Days 2, 5, and 7); in contrast, in BC-PCs and AP-PCs, PLT apoptosis and activation have approximately equal sensitivity to storage (Table 2, Columns 3 and 4, 18.6%-21.3% vs. 20%-25% for Day 7). Furthermore, PLT apoptosis in PRPPCs is more resistant to storage than is apoptosis in BC-PCs and AP-PCs (Tables 1 and 2A, Columns 2-4, p < 0.01 for Days 5 and 7); in contrast, activation in PRPPCs is more sensitive to storage than is activation in BC-PCs and AP-PCs (Table 2B, Columns 2-4, 65.8 9.7% vs. 20%-25% for Day 7). In conclusion, the analysis of these recently reported data indicates that PLT activation rather than apoptosis most contributes to the PLT storage lesion during storage of PRP-PCs for 7 days. Therefore, PLT activation, and not apoptosis, should be the main concern in maintaining quality of PRP-PCs during blood banking storage. In contrast, for BC-PCs and AP-PCs, both activation and

Mitochondrial Inner Membrane Depolarization as a Marker of Platelet Apoptosis : Disclosure of Nonapoptotic Membrane Depolarization

Availability of universal marker for the diagnosis of platelet apoptosis is an important but currently unresolved goal of platelet physiology investigations. Mitochondrial inner transmembrane potential (▵Ψm) depolarization is frequently used as a marker of apoptosis in nucleated cells and anucleate platelets. Since ▵Ψm depolarization in platelets is also frequently associated with concurrent induction of other apoptotic responses, it may appear that ▵Ψm depolarization is a good universal marker of platelet apoptosis. However, data presented in the current study indicate that this is incorrect. We report here fundamental differences in the effects of potassium ionophore valinomycin and calcium ionophore A23187 on human platelet apoptosis. Although both A23187-triggered and valinomycin-triggered ▵Ψm depolarization are strongly induced, the former is dependent on the opening of mitochondrial permeability transition pore (MPTP) and the latter is MPTP-independent. Furthermore, effects of calcium and potassium ionophores on other apoptotic events are also basically different. A23187 induces caspase-3 activation, proapoptotic Bax and Bak protein expression, phosphatidylserine exposure, and microparticle formation, whereas valinomycin does not induce these apoptotic manifestations. Discovery of targeted ▵Ψm depolarization not associated with apoptosis in valinomycin-treated platelets indicates that this marker should not be used as a single universal marker of platelet apoptosis in unknown experimental and clinical settings as it may lead to a false-positive apoptosis diagnosis.