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The in vitro thromboxane A2 production by platelets of diabetic patients is normal at physiological [Ca2+]
Vol. 65, Suppl. 1
ABSTRACTS OF 12TH INTNAT’L CONGRESS
s37
C69 THE IN VITRO THROMBOXANE A2 PRODUCTIONBY PLATELETS OF DIABETIC PATIENTS IS NORMALAT PHYSIOLOGICAL [~a~+].C.R. Falcon, M. Cattaneo, A. Ghidoni*, P.M. Mannucci. A. Bianchi Bonomi Hemophilia and Thrombosis Center and *lst Clinical Medicine. University of Milan, Italy. Platelets of patients with DM produce normal amounts of thromboxane (TX) A in viva, whereas they usually show increased aggregability and TxB p?.oduction in vitro. Since in vitro studies are usually performed i$ titrated PRP, we tested whether the increased TXA production observed in DM platelets is due to the potentiating effect of me%-la with low [Ca2+l. Twenty patients with IDDM, 20 with NIDDM and 37 controls were studied. Blood was anticoagulated with 12.9mM triszdium citrate, 0.7mM PPACK or both. Platelet aggregation (PA), release of C-serotonin (5I-W) and TXB production were induced
by ADP 2-4
uM, adrenaline
(AA) 0.7 mM. Results. production (p
5 uM,
collagen
2 ug/ml
o? arachidonic
1) PRP in citrate. AA-induced PA (p
and
acid TXB
No othe?
differences were found. 2) PRP in PPACK. No differences between DM patients and controls were found. 3) PRP in PPACK + citrate. Results similar to those obtained with citrate were obtained. In conclusion, the artifactual potentiation of TXB production in media with low [Ca2”] accounts for the increased generation20f TxJ3 by DM platelets found in in vitro studies. DM platelets produce normal am&nts of TxA2 in vivo and in vitro.
c70 MAYTHEREDUCEDMEMBRANE LlPlDFLUlDlTYUNDERLlETHEDECREASEDAMOUNTOFGPllb AND GPlIla IN PLATELETS FROM DIABETIC JUVENILES? C. Watala’, T. Pietruchal, B. Walkowiak’, E. Czemiawska2 and C.S. Ciemiewski’ ‘Department of Biophysics and 2 Institute of Paediatrics, Medical School of L&z, PL The reduced membrane fluidity in platelet membranes from diabetic subjects, attributed to the enhanced glpation of membrane proteins, was reported to be associated with platelet hypersensitivity to thrombin. It is also known that the modulation in membrane lipid fluidity may determine the degree of accessibility of membrane receptors and their response to fluidity changes. In order to examine whether the alterations in cell membrane lipid bilayer dynamics are somehow related to the amount platelet membrane glycoproteins GPlIb and GPllla, parameters, we compared 12 diabeticjuvenilesubjects with 12 age-andsex-matchedcontrols.Themeansteady-statefluorescence polarization values (pDPH) in I ,6-diphenyl- 1,3,5-hexatriene-labeled isolated platelet membranes from diabetic subjects were significantly greater than from control subjects (0.23 1 + 0.011 vs 0.222 + 0.008, p < 0.02), thus indicating for the reduced membrane lipid fluidity in diabetic platelets. These changes were inversely correlated with the amounts of platelet membrane glycoprotein GPllb (r--0.624, p c 0.001) and GPllla (r--0.557, p < 0.001). The amounts of these glycoproteins were significantly reduced in diabetic subjects (GPIIB: 1.65 + 0.16 pg/ IO* pl vs 0.93 kO.14 pg/lO* pl, ~~0.02; GPllla: 2.15k0.29 vs 1.47k0.22, ~~0.02). We did not revealany significant relation among pDPn and either the glycated haemoglobin (HbA,=) or clotting time (CT) and bleeding time, although
the levelsofHbA,,differed significantlyin both groups(4.8 ~0.4 in controlsvs 9.7 22.3 in diabetics).Relyingon the existing relationship between membrane lipid fluidity and the apparent exposure of membrane proteins, we concluded that the alterations in membrane fluidity may be responsible for hypersensitivity of diabetic platelets. In accord with that, the diminished amount of GPllb and GPllla might result from the accelerated membrane protein shedding in diabetic platelets.
ABSTRACTS OF 12TH INTNAT’L CONGRESS
s37
C69 THE IN VITRO THROMBOXANE A2 PRODUCTIONBY PLATELETS OF DIABETIC PATIENTS IS NORMALAT PHYSIOLOGICAL [~a~+].C.R. Falcon, M. Cattaneo, A. Ghidoni*, P.M. Mannucci. A. Bianchi Bonomi Hemophilia and Thrombosis Center and *lst Clinical Medicine. University of Milan, Italy. Platelets of patients with DM produce normal amounts of thromboxane (TX) A in viva, whereas they usually show increased aggregability and TxB p?.oduction in vitro. Since in vitro studies are usually performed i$ titrated PRP, we tested whether the increased TXA production observed in DM platelets is due to the potentiating effect of me%-la with low [Ca2+l. Twenty patients with IDDM, 20 with NIDDM and 37 controls were studied. Blood was anticoagulated with 12.9mM triszdium citrate, 0.7mM PPACK or both. Platelet aggregation (PA), release of C-serotonin (5I-W) and TXB production were induced
by ADP 2-4
uM, adrenaline
(AA) 0.7 mM. Results. production (p
5 uM,
collagen
2 ug/ml
o? arachidonic
1) PRP in citrate. AA-induced PA (p
and
acid TXB
No othe?
differences were found. 2) PRP in PPACK. No differences between DM patients and controls were found. 3) PRP in PPACK + citrate. Results similar to those obtained with citrate were obtained. In conclusion, the artifactual potentiation of TXB production in media with low [Ca2”] accounts for the increased generation20f TxJ3 by DM platelets found in in vitro studies. DM platelets produce normal am&nts of TxA2 in vivo and in vitro.
c70 MAYTHEREDUCEDMEMBRANE LlPlDFLUlDlTYUNDERLlETHEDECREASEDAMOUNTOFGPllb AND GPlIla IN PLATELETS FROM DIABETIC JUVENILES? C. Watala’, T. Pietruchal, B. Walkowiak’, E. Czemiawska2 and C.S. Ciemiewski’ ‘Department of Biophysics and 2 Institute of Paediatrics, Medical School of L&z, PL The reduced membrane fluidity in platelet membranes from diabetic subjects, attributed to the enhanced glpation of membrane proteins, was reported to be associated with platelet hypersensitivity to thrombin. It is also known that the modulation in membrane lipid fluidity may determine the degree of accessibility of membrane receptors and their response to fluidity changes. In order to examine whether the alterations in cell membrane lipid bilayer dynamics are somehow related to the amount platelet membrane glycoproteins GPlIb and GPllla, parameters, we compared 12 diabeticjuvenilesubjects with 12 age-andsex-matchedcontrols.Themeansteady-statefluorescence polarization values (pDPH) in I ,6-diphenyl- 1,3,5-hexatriene-labeled isolated platelet membranes from diabetic subjects were significantly greater than from control subjects (0.23 1 + 0.011 vs 0.222 + 0.008, p < 0.02), thus indicating for the reduced membrane lipid fluidity in diabetic platelets. These changes were inversely correlated with the amounts of platelet membrane glycoprotein GPllb (r--0.624, p c 0.001) and GPllla (r--0.557, p < 0.001). The amounts of these glycoproteins were significantly reduced in diabetic subjects (GPIIB: 1.65 + 0.16 pg/ IO* pl vs 0.93 kO.14 pg/lO* pl, ~~0.02; GPllla: 2.15k0.29 vs 1.47k0.22, ~~0.02). We did not revealany significant relation among pDPn and either the glycated haemoglobin (HbA,=) or clotting time (CT) and bleeding time, although
the levelsofHbA,,differed significantlyin both groups(4.8 ~0.4 in controlsvs 9.7 22.3 in diabetics).Relyingon the existing relationship between membrane lipid fluidity and the apparent exposure of membrane proteins, we concluded that the alterations in membrane fluidity may be responsible for hypersensitivity of diabetic platelets. In accord with that, the diminished amount of GPllb and GPllla might result from the accelerated membrane protein shedding in diabetic platelets.