Increased expression of tissue factor and receptor for advanced glycation end products in peripheral blood mononuclear cells of patients with type 2 diabetes mellitus with vascular complications.

The aim of the study was to determine the correlation between the expression of tissue factor (TF) and the receptor for advanced glycation end products (RAGEs) and vascular complications in patients with longstanding uncontrolled type 2 diabetes (T2D). TF and RAGE mRNAs as well as TF antigen and activity were investigated in 21 T2D patients with and without vascular complications. mRNA expression was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) in nonstimulated and advanced glycation end product (AGE) albumin-stimulated peripheral blood mononuclear cells (PBMCs). TF antigen expression was determined by enzyme-linked immunosorbent assay (ELISA) and TF activity by a modified prothrombin time assay. Basal RAGE mRNA expression was 0.2 +/- 0.06 in patients with complications and 0.05 +/- 0.06 patients without complications (P =.004). Stimulation did not cause any further increase in either group. TF mRNA was 0.58 +/- 0.29 in patients with complications and 0.21 +/- 0.18 in patients without complications (P =.003). Stimulation resulted in a nonsignificant increase in both groups. Basal TF activity (U/10(6) PBMCs) was 18.4 +/- 13.2 in patients with complications and 6.96 +/- 5.2 in patients without complications (P =.003). It increased 3-fold in both groups after stimulation (P =.001). TF antigen (pg/10(6) PBMCs) was 33.7 +/- 28.6 in patients with complications, 10.4 +/- 7.8 in patients without complications (P =.02). Stimulation tripled TF antigen in both groups of patients (P =.001). The RAGE/TF axis is up-regulated in T2D patients with vascular complications as compared to patients without complications. This suggests a role for this axis in the pathogenesis of vascular complications in T2D.

Original Article: Insensitivity of Platelet Aggregation to Inhibition by Prostaglandin I(2) (PGI(2)) and Prostaglandin E(1) (PGE(1)), and Reduced Platelet c-AMP Accumulation in Response to PGE(1) in Insulin-dependent Diabetic Patients.

In insulin-dependent diabetics (IDDM) and peripheral occlusive arterial disease (POAD), inhibition of platelet aggregation (PA) by PGI(2) and PGE(1) was reduced (p <0.02) compared with non-diabetics and POAD as well as with healthy subjects at a similar extent of PA (in the 3 groups). c-AMP content in resting platelets of IDDM and chronic renal failure was decreased (mean ± SD in pmo1/2.5 × 10(8) platelets): 5.3 ± 1.1 compared with the content in healthy subjects: 8.0 ± 1.12 and P< 0.012. Moreover, c-AMP content in PGE(1)-stimulated platelets was also reduced in these patients. Our results indicate that diabetic patients are characteristically less sensitive to inhibition by the 2 prostanoids, presumably through attenuated activity of the c-AMP generating system.

Increased Thromboxane B(2) Generation and ß-Thromboglobulin Release from Activated Platelets in Myotonic Dystrophy.

Compared to normal controls, platelets from myotonic dystrophy (MyD) patients, released significantly more ß-thromboglobulin (ßTG) and generated more thromboxane B(2) (TXB(2)) when platelet aggregation was induced by ADP (1.5 µM) or epinephrine 1 µM. TXB(2) generation in MyD platelets at 1 and 3 min was positively correlated (p < 0.05) to platelet aggregation (PA). This suggests that, 1 min after the addition of the PA inducers, the platelets of MyD patients are already at the second (irreversible) phase of PA. Together with our previous data, these results may indicate hyperactivity of platelets caused by a membrane abnormality in MyD.

An abnormal pattern of multiple platelet function abnormalities and increased thromboxane generation in patients with primary thrombocytosis and thrombotic complications.

Platelet aggregation (PA) induced by ADP, collagen and epinephrine, plasma levels of beta-thromboglobulin (beta TG) and thromboxane B2 (TXB2) and serum TXB2 generation were studied in 11 patients with primary thrombocytosis (7 with essential thrombocythaemia and 4 with polycythaemia vera) and compared with 16 healthy subjects. 5 patients suffered from peripheral vascular ischaemia and another 3 had venous thrombosis, but none had bleeding complications. The patients showed an abnormal pattern of platelet function and of thromboxane generation distinct from the healthy subjects in three aspects. a) Shape change was 5-26 times greater, the lag-time of collagen PA was 2.3-2.9 times longer and the extent of epinephrine PA was nil or very low. ADP- or collagen-induced PA was also reduced (p less than 0.02). b) Plasma TXB2 generation (corrected to a normal platelet concentration) stimulated by the three PA inducers was within the range of the healthy subjects in spite of the reduced extent of PA. c) Plasma beta TG level and serum TXB2 generation (both corrected to a normal platelet concentration) were 2.9-7.1 times higher (p less than 0.001) indicating enhanced in vivo platelet activation and possibly increased thrombin generation. These abnormalities were not detected in another 4 patients with secondary thrombocytosis. The abnormal pattern of platelet function and thromboxane generation can be a useful laboratory method in the evaluation of patients with primary thrombocytosis. It might also explain the thrombotic complications which occurred in 8 of the patients in a manner such that increased or normal TXB2 generation overcomes the reduced extent of PA. In this respect, the pronounced serum TXB2 synthesis might be a marker of intravascular thrombosis.

Abnormal typical pattern of platelet function and thromboxane generation in unstable angina.

Platelet aggregation (PA), platelet thromboxane B2 (TXB2) generation and 14C 5-hydroxytryptamine (5HT) release were studied in 13 patients with unstable angina, and compared to 14 patients with stable angina and 16 healthy controls. A typical pattern, distinct in 4 aspects from stable angina patients or controls, was observed in the unstable angina patients. ADP or collagen induced shape change was 3-4 times greater, the extent of epinephrine induced PA was nil or very low, the extent of collagen induced 14C 5HT release was also reduced while collagen induced platelet TXB2 generation was increased in spite of a reduced extent of PA. The extent of ADP or collagen induced PA was also significantly reduced. These results indicate a platelet membrane abnormality occurring presumably during contact of the circulating platelets with a non-occlusive thrombus observed at sites of ruptured plaques in unstable angina patients. Since also the pattern (20-30% overlap with control values) was distinct from that of stable angina patients, it might indicate an active thrombotic process. Plasma beta-thromboglobulin (beta TG) and TXB2 levels and serum TXB2 generation were also studied in the cardiac patients and controls and in another 10 patients with advanced peripheral occlusive arterial disease (POAD). Plasma beta TG and TXB2 levels were slightly elevated in the unstable angina patients and markedly elevated in the POAD patients. Serum TXB2 generation was, however, elevated in the stable angina patients (p less than 0.002) and more so in the unstable angina patients (p less than 0.001) compared to controls or to POAD patients. This was presumably mediated through enhanced thrombin generation. These results suggest that the measured plasma beta TG variable in the unstable angina patients is not useful in the assessment of in vivo platelet activation. It is presumably reflecting the sum of local enhanced platelet activation (at sites of ruptured plaques) and of reduced function of the "defective" circulating platelets. The ability of the platelets of unstable angina patients to generate large amounts of TXB2 if occurring in vivo might induce an intense coronary vasospasm.

Increased platelet aggregation and release reaction in myotonic dystrophy.

Platelet aggregation (PA) induced by (-)-epinephrine and adenosine diphosphate (ADP) was studied in 16 patients with myotonic dystrophy (MyD) and 14 healthy subjects. Plasma beta-thromboglobulin level (beta-TG), a useful marker of in vivo platelet release reaction, as well as in vitro 5-[14C]hydroxytryptamine (5-HT) release, were also studied. The extent of PA induced by (-)-epinephrine at 1 and 3 min and by ADP at 3 min was significantly higher in the patients than in controls. Plasma beta-TG and ADP- or epinephrine-induced platelet 5-HT release were also increased in MyD patients. These results suggest that an abnormality in release as well as in alpha 2-receptor functioning occurs in the platelets of MyD patients. The relation of this abnormality to changes in Ca2+ fluxes through the platelet membrane is discussed.

Effect of heparin and dihydroergotamine on platelet adenosine-3',5'-cyclic monophosphate.

Human platelet adenosine-3',5'-cyclic monophosphate (cAMP) levels were determined in platelet rich plasma (PRP) and in washed platelets by a modification of the protein binding assay; the validation of the method is described. Dihydroergotamine (DHE) inhibited epinephrine induced platelet aggregation (ID50 = 2.5 X 10(-7) mol/l), and increased cAMP levels in platelets by an alpha-adrenergic receptor blocking effect, since phentolamine but not propranolol, behaved similarly. The DHE induced cAMP accumulation was correlated to the inhibitory effect on aggregation and showed a characteristic alpha-adrenergic receptor pattern in the presence of alprostadil (PGE1) and epinephrine but not collagen or adenosine diphosphate (ADP). Thrombin induced aggregation was similarly affected by DHE but with 100 times higher concentration. Heparin was found to increase slightly ADP and epinephrine induced aggregation and to decrease cAMP. Also, heparin was found to inhibit thrombin induced platelet aggregation. In washed platelets, the inhibitory effect of thrombin on PGE1 induced cAMP accumulation was counteracted by heparin. This indicates that the binding site of thrombin on platelets is important in the control of adenyl cyclase. Evidence is presented that some of the beneficial synergistic effect of DHE and heparin may consist in the ability of those compounds to produce opposite effects on cAMP system in platelets.

Enhanced platelet release reaction, shortened platelet survival time and increased platelet aggregation and plasma thromboxane B2 in chronic obstructive arterial disease.

Plasma beta-thromboglobulin (beta TG) and thromboxane B2 (TXB2) level, platelet aggregation (PA) in platelet rich plasma, platelet survival time using 111Indium radiolabelled platelets and platelet sensitivity to prostacyclin (PGI2) were measured in chronic obstructive arterial disease (COAD) patients. Severity of the disease was assessed by the ankle pressure index using Doppler auscultation. Platelet survival time was shorter, plasma beta TG and TXB2 and the rate and extent of PA induced by ADP or 1-epinephrine (but not collagen) were greater in the patients than in controls. Beta TG was inversly correlated with the pressure index and positively with TXB2 indicating increased platelet TXA2 synthesis. Platelet sensitivity to PGI2 was similar in the patients and controls. These results indicate increased platelet consumption and enhanced in vivo platelet activation and PA in COAD. The enhanced activation correlates with the severity of the disease and the activated platelets presumably synthesize increased amounts of TXA2. It is therefore concluded that platelets might be involved in the pathogenesis of COAD. The normal platelet sensitivity to PGI2 suggests that the administration of the compound may improve the abnormal platelet function in COAD patients and may attenuate the progression of the disease.

Effect of adenyl-cyclase activators, phosphodiesterase inhibitors and pyridoxal-5-phosphate on platelet aggregation and adenosine-3'-5'-cyclic monophosphate accumulation.

The effect of prostaglandin E1 (PGE1) and compound BW245C, adenyl-cyclase activators, theophylline, papaverine and dipyridamole, phosphodiesterase (PDE) inhibitors and pyridoxal-5-phosphate (PALP) on inhibition of platelet aggregation (PA) and platelet c-AMP accumulation was determined in human platelet-rich plasma (PRP) and washed platelets. PGE1 at 280 nM and BW245C at 7.7 nM induced a significant PA inhibition in PRP and washed platelets (though less pronounced by PGE1) concomitant to a very large increase (8-13-fold) in platelet c-AMP level both in PRP and washed platelets. At comparable PA inhibition, c-AMP level was not significantly changed by PALP and only moderately (but significantly) increased (2-4.6-fold) by PDE inhibitors. PALP or theophylline potentiated both PGE1-induced platelet c-AMP accumulation and PA inhibition. Yet PALP potentiated theophylline-induced PA inhibition without affecting platelet c-AMP level. Our results indicate that 2 c-AMP pools are presumably present in the platelets, since at comparable c-AMP accumulation PDE inhibitors or BW245C were more effective than PGE1 in PA inhibition in PRP. Moreover, this pattern was more pronounced in washed platelets and was also found in the presence of thrombin and adenosine diphosphate which induced a decrease in platelet c-AMP level. The effect of PALP on PA inhibition is presumably mediated by 2 mechanisms: a) a direct effect on the platelet membrane independent from the c-AMP system. b) In the presence of PGE1 the increment in PA inhibition, is through further indirect activation of the adenyl-cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of pyridoxal-5-phosphate on the inhibition of platelet aggregation and adenosine-3'-5'-cyclic monophosphate accumulation in human platelets.

Platelet adenosine-3'-5'-cyclic monophosphate (c-AMP) was determined in platelet rich plasma and washed platelets by a modification of the c-AMP protein binding assay. Studies were performed in the presence and absence of platelet aggregation inducers (adenosine diphosphate and thrombin) and inhibitors (pyridoxal-5-phosphate (PALP), prostaglandin E1 (PGE1), theophylline and papaverine). At 70% inhibitory concentration of platelet aggregation (PA) induced by papaverine or theophylline, a small but significant increase in platelet c-AMP level was found. With PALP however the inhibition of PA was not associated with a significant increase in the c-AMP level. PALP or theophylline potentiated greatly both the inhibition of PA and c-AMP accumulation induced by PGE1. Yet PALP potentiated the inhibition of PA induced by theophylline without increasing platelet c-AMP level. Our results indicate that the inhibition of PA by PALP is not mediated by c-AMP accumulation. However in the presence of PGE1, the increment in PA inhibition, is mediated by further indirect activation of the adenyl-cyclase.