STIMULATED GLANZMANNS-THROMBASTHENIA PLATELETS PRODUCE MICROVESICLES - MICROVESICULATION CORRELATES BETTER TO EXPOSURE OF PROCOAGULANT SURFACE THAN TO ACTIVATION OF GPIIB-IIIA

The mechanism of formation of platelet-derived microvesicles remains c ontroversial. The aim of the present work was to study the formation o f microvesicles in view of a possible involvement of the GPIIb-IIIa co mplex, and of exposure of negatively charged phospholipids as procoagu lant material on the platelet surface. This was studied in blood from three Glanzmann's thrombasthenia patients lacking GPIIb-IIIa and healt hy blood donors. MAb FN52 against CD9 which activates the complement s ystem and produces microvesicles due to a membrane permeabilization, A DP (9.37 mu M), and the thrombin receptor agonist peptide SFLLRN (100 mu M) that activates platelets via G-proteins were used as inducers. I n a series of experiments platelets were also preincubated with PGE(1) (20 mu M). The number of liberated microvesicles, as per cent of the total number of particles (including platelets), was measured using fl ow cytometry with FITC conjugated antibodies against GPIIIa or GPIb, A ctivation of GPIIb-IIIa was detected as binding of PAC-1, and exposure of aminophospholipids as binding of annexin V. With normal donors, ac tivation of the complement system induced a reversible PAC-1 binding d uring shape change. A massive binding of annexin V was seen during sha pe change as an irreversible process, as well as formation of large nu mbers of microvesicles (60.6 +/- 2.7%) which continued after reversal of the PAC-1 binding. Preincubation with PGE(1) did not prevent bindin g of annexin V, nor formation of microvesicles (49.5 +/- 2.7%), but ab olished shape change and PAC-1 binding after complement activation. Th rombasthenic platelets behaved like normal platelets after activation of complement except for lack of PAC-1 binding (also with regard to th e effect of PGE(1) and microvesicle formation). Stimulation of normal platelets with 100 mu M SFLLRN gave 16.3 +/- 1.2% microvesicles, and s trong PAC-1 and annexin V binding. After preincubation with PGE(1) nei ther PAC-1 nor annexin V binding, nor any significant amount of microv esicles could be detected. SFLLRN activation of the thrombasthenic pla telets produced a small but significant number of microvesicles (6.4 /- 0.8%). Incubation of thrombasthenic platelets with SFLLRN after pre incubation with PGE(1), gave results identical to those of normal plat elets. ADP activation of normal platelets gave PAC-1 binding, but no s ignificant annexin V labelling, nor production of microvesicles. Thus, different inducers of the shedding of microvesicles seem to act by di fferent mechanisms. For al inducers there was a strong correlation bet ween the exposure of procoagulant surface and formation of microvesicl es, 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 afte r activation of the complement system, but seems to be of importance, but not absolutely required, after stimulation with SFLLRN.