Summary
The mechanism of formation of platelet-derived microvesicles remains controversial.
The aim of the present work was to study the formation of microvesicles in view of
a possible involvement of the GPIIb-IIIa complex, and of exposure of negatively charged
phospholipids as procoagulant material on the platelet surface. This was studied in
blood from three Glanzmann’s thrombasthenia patients lacking GPIIb-IIIa and healthy
blood donors. MAb FN52 against CD9 which activates the complement system and produces
microvesicles due to a membrane permeabilization, ADP (9.37 μM), and the thrombin
receptor agonist peptide SFLLRN (100 μM) that activates platelets via G-proteins were
used as inducers. In a series of experiments platelets were also preincubated with
PGE1 (20 μM). The number of liberated microvesicles, as per cent of the total number of
particles (including platelets), was measured using flow cytometry with FITC conjugated
antibodies against GPIIIa or GPIb. Activation of GPIIb-IIIa was detected as binding
of PAC-1, and exposure of aminophospholipids as binding of annexin V. With normal
donors, activation of the complement system induced a reversible PAC-1 binding during
shape change. A massive binding of annexin V was seen during shape change as an irreversible
process, as well as formation of large numbers of microvesicles (60.6 ±2.7%) which
continued after reversal of the PAC-1 binding. Preincubation with PGE1 did not prevent binding of annexin V, nor formation of microvesicles (49.5 ± 2.7%),
but abolished shape change and PAC-1 binding after complement activation. Thrombasthenic
platelets behaved like normal platelets after activation of complement except for
lack of PAC-1 binding (also with regard to the effect of PGE1 and microvesicle formation). Stimulation of normal platelets with 100 μM SFLLRN gave
16.3 ± 1.2% microvesicles, and strong PAC-1 and annexin V binding. After preincubation
with PGE1 neither PAC-1 nor annexin V binding, nor any significant amount of microvesicles
could be detected. SFLLRN activation of the thrombasthenic platelets produced a small
but significant number of microvesicles (6.4 ± 0.8%). Incubation of thrombasthenic
platelets with SFLLRN after preincubation with PGE1, gave results identical to those of normal platelets. ADP activation of normal platelets
gave PAC-1 binding, but no significant annexin V labelling, nor production of microvesicles.
Thus, different inducers of the shedding of microvesicles seem to act by different
mechanisms. For all inducers there was a strong correlation between the exposure of
procoagulant surface and formation of microvesicles, suggesting that the mechanism
of microvesicle formation is linked to the exposure of aminophospholipids. The results
also show that the GPIIb-IIIa complex is not required for formation of microvesicles
after activation of the complement system, but seems to be of importance, but not
absolutely required, after stimulation with SFLLRN.
