Insulin directly reduces platelet sensitivity to aggregating agents. Studies in vitro and in vivo.

The aim of this study was to investigate the influence of insulin on platelet function, both in vitro and in vivo. For the in vitro investigation, we evaluated whether insulin affects platelet function at a physiological hormone concentration by incubating the platelet-rich plasma (PRP) of fasting subjects with human regular insulin at the final concentration of 40 microU/ml for 30 min; we observed a significant reduction of platelet sensitivity to all the aggregating agents employed, i.e., ADP, platelet-activating factor (PAF), epinephrine, collagen, and Na+ arachidonate. To investigate whether the insulin effect on platelets is dose dependent, we incubated the PRP of fasting subjects with different concentrations of human regular insulin (40, 80, 120, and 160 microU/ml) for 5 min, and we observed that the insulin-induced reduction of platelet sensitivity to aggregating agents is a dose-dependent phenomenon. Furthermore, the comparison between the platelet responses after 5 and 30 min of incubation with insulin showed that the insulin effect on platelet aggregation is time dependent. The lack of specificity of its inhibiting activity suggests that insulin does not interfere with the initial binding of each aggregating agent at specific sites but does influence a common step of platelet aggregation. Our study rules out the possibility that insulin reduces platelet-function-modifying intraplatelet cAMP levels or thromboxane A2 production, because this hormone decreases the platelet concentrations of cAMP--a phenomenon that, per se, promotes platelet aggregation--and does not modify collagen or Na+ arachidonate--induced platelet production of thromboxane A2, measured by radioimmunoassay of its stable-metabolite thromboxane B2.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin increases guanosine-3',5'-cyclic monophosphate in human platelets. A mechanism involved in the insulin anti-aggregating effect.

To investigate whether insulin reduces platelet aggregability through a modulation of the guanosine-3',5'-cyclic monophosphate (cGMP) concentrations, we determined by a radioimmunoassay the cGMP values in the platelet-rich plasma (PRP) obtained from 17 healthy volunteers and incubated for 3 min with different concentrations of human recombinant insulin (0, 240, 480, 720, 960, and 1,920 pM). Insulin induced a dose-dependent cGMP increase, from 18.5 +/- 3.3 to 42.0 +/- 6.4 pmol/10(9) platelets (P = 0.0001). This increase was completely blunted when PRP was preincubated for 20 min with the tyrosine kinase inhibitor genistein (10 microM) or with the guanylate cyclase inhibitor methylene blue (10 microM), but the increase remained highly significant (P = 0.003 and 0.009) when PRP was preincubated for 20 min with the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX, 500 microM) or with the nitric oxide synthase inhibitor NG-mono-methyl-L-arginine (L-NMMA, 30 microM). Finally, the insulin-induced decrease of platelet aggregability to collagen and ADP was completely blunted when PRP was preincubated with 10 microM of the guanylate cyclase inhibitor methylene blue. This study demonstrates that the platelet anti-aggregatory effect exerted by insulin is attributable to the insulin-induced increase of cGMP that is due to a direct receptor-mediated platelet guanylate cyclase activation.

The insulin-induced increase of guanosine-3',5'-cyclic monophosphate in human platelets is mediated by nitric oxide.

To investigate whether the insulin-induced increase of guanosine-3',5'-cyclic monophosphate (cGMP) in human platelets is mediated by nitric oxide or is influenced by the nitric oxide precursor L-arginine, we measured cGMP in platelet-rich plasma obtained from healthy volunteers incubated for 3 min with human recombinant insulin (0, 240, 480, 960, and 1,920 pmol/l) both with and without 1) a 20-min incubation with the nitric oxide-synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA) (50, 70, 100, and 1,000 micromol/l; n = 5 for each dose) and 2) a 20-min incubation with the nitric oxide precursor L-arginine (300 micromol/l; n = 6). In a first set of experiments, insulin induced a dose-dependent cGMP increase, from 9.8 +/- 0.8 to 45.6 +/- 5.5 pmol/10(9) platelets (P = 0.0001); in the presence of 1 mmol/l L-NMMA, this increase was blunted, cGMP being 8.9 +/- 1.4 and 11.1 +/- 2.2 pmol/10(9) platelets at 0 and 1,920 pmol/l insulin, respectively (NS). In the experiments with 70 and 100 micromol/l L-NMMA, the insulin effect on cGMP was inhibited, whereas 50 micromol/l L-NMMA did not blunt this insulin effect. In another set of experiments carried out to investigate the effects of L-arginine, insulin induced a dose-dependent cGMP increase, from 23.6 +/- 6.9 to 59.0 +/- 12.0 pmol/10(9) platelets (P = 0.0001); with L-arginine, basal cGMP values increased to 35.5 +/- 6.6 pmol/10(9) platelets (P = 0.05), and insulin maintained its ability to enhance dose-dependently cGMP values, which rose to 76.8 +/- 19.4 pmol/10(9) platelets (P = 0.003). This study carried out in human platelets demonstrates that the cGMP increase induced by insulin, which accounts for the antiaggregating effect of the hormone, is mediated by nitric oxide.

Insulin stimulates nitric oxide synthesis in human platelets and, through nitric oxide, increases platelet concentrations of both guanosine-3', 5'-cyclic monophosphate and adenosine-3', 5'-cyclic monophosphate.

The insulin-induced platelet anti-aggregating effect is attributed to a nitric oxide (NO)-mediated increase of cyclic guanosine monophosphate (cGMP). The aim of this work, carried out in human platelets, is to show whether insulin increases NO synthesis in platelets and whether it enhances not only cGMP but also cyclic adenosine monophosphate (cAMP) in these cells. We observed that 1) insulin dose-dependently increases NO production, evaluated as citrulline synthesis from L-arginine (n = 4, P = 0.015); 2) insulin dose-dependently increases not only cGMP but also cAMP: for instance, after 8 min of insulin incubation at 1,920 pmol/l, cAMP increased from 39.8 +/- 1.4 to 121.3 +/- 12.6 pmol/10(9) platelets (n = 16, P = 0.0001); 3) when insulin is incubated for 120 min, the increase of cGMP and cAMP shows a plateau between 2 and 20 min, and while the effect on cGMP is significant until 120 min, the effect on cAMP is no more significant at 60 and 120 min; 4) insulin increases the effects on cAMP of the adenylate cyclase agonists Iloprost and forskolin (n = 5, P = 0.0001) and enhances their platelet anti-aggregating effects (n = 6 and 8, respectively; P = 0.0001); and 5) the inhibition of NO synthase by N(G)-monomethyl-L-arginine blunts both the insulin effects on basal cGMP and cAMP (n = 4) and those on the Iloprost- and forskolin-induced cAMP increase (n = 5). Thus, insulin increases NO synthesis in human platelets, and, through NO, enhances both cGMP and cAMP. The platelet anti-aggregating effect exerted by insulin is, therefore, a NO-mediated phenomenon involving both cGMP and cAMP.

Studies on mechanisms involved in hypoglycemia-induced platelet activation.

The aim of our study was to investigate the mechanisms involved in hypoglycemia-induced platelet activation. Sixteen healthy male subjects received a 60-min intravenous infusion of human regular insulin at the rate of 64 mU . m-2 . min-1: throughout 150 min, we serially measured plasma concentrations of glucose, insulin, and counterregulatory hormones; platelet sensitivity to ADP, thrombin and platelet-activating factor; plasma concentrations of platelet markers for specific proteins of in vivo release reaction (beta-thromboglobulin and platelet factor 4). Our study showed that insulin-induced hypoglycemia causes a significant increase in platelet sensitivity to aggregating agents in vitro and a platelet release reaction in vivo. Hypoglycemia-induced platelet activation was not correlated with plasma glucose concentrations at nadir and occurred before the increase of plasma growth hormone and cortisol. To further elucidate the mechanisms of hypoglycemia-induced platelet activation, we incubated in vitro platelet-rich plasma (PRP) of seven fasting healthy subjects with the same concentrations of insulin, epinephrine, glucagon, growth hormone, and cortisol measured in vivo during insulin-induced hypoglycemia. Only epinephrine was able to increase platelet sensitivity to aggregating agents. To investigate the role of alpha-adrenergic receptors in this phenomenon, we also studied four healthy subjects on another occasion, repeating the above-described insulin infusion together with intravenous infusion of phentolamine (-15 to +150 min), 5 mg over 2 min followed by 500 micrograms/min. alpha-Blockade was able to suppress hypoglycemia-induced increase of platelet sensitivity to aggregating agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Impaired insulin-induced platelet antiaggregating effect in obesity and in obese NIDDM patients.

To investigate the effects of insulin on platelets in obesity and in non-insulin-dependent diabetes mellitus (NIDDM)--classic insulin-resistant states--we determined ADP-induced platelet aggregation and platelet cGMP (guanosine 3',5'-cyclic monophosphate) content in platelet-rich plasma obtained from nine obese subjects and nine age-matched healthy volunteers and from eight NIDDM obese patients and nine age-matched healthy volunteers after a 3-min incubation with human recombinant insulin (0, 240, 480, 960, and 1,920 pmol/l). Platelet aggregation was evaluated using different ADP doses to measure the ADP concentration determined on the basis of a dose-response curve necessary to elicit a maximal aggregation of 50% (ED50). Insulin induced a dose-dependent decrease of platelet aggregation to ADP (P = 0.0001) in healthy subjects. A significant effect was evident starting from an insulin concentration of 240 pmol/l. On the contrary, in insulin-resistant subjects, insulin reduced platelet sensitivity to ADP only at a concentration of 1,920 pmol/l. When ADP ED50 values obtained in platelet-rich plasma incubated with insulin were expressed in percentage of the ADP ED50 values obtained in platelet-rich plasma without insulin, considered as 100%, we observed that ADP ED50 with 1,920 pmol/l insulin was 153.6 +/- 13.2% in the younger healthy subject group (P = 0.004), 150.0 +/- 3.8% in the older healthy subject group (P = 0.0001), 116.1 +/- 6.1% in obese subjects (P = 0.031), and 120.0 +/- 8.6% in NIDDM patients (P = 0.05). In healthy subjects, insulin induced a dose-dependent increase of platelet cGMP (P = 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Postprandial exercise in type I diabetic patients on multiple daily insulin injection regimen.

This study shows the influence on plasma glucose concentrations of 45 min of mild exercise (48 +/- 4% of maximum aerobic capacity) performed 180 min after breakfast and 195 min after a subcutaneous injection of regular insulin by six type I (insulin-dependent) diabetic patients on a three-daily insulin injection regimen (regular insulin before breakfast and lunch, regular + intermediate insulin before supper). It has been observed that such exercise does not induce a large plasma glucose decrease. Actually, plasma glucose concentrations were 99 +/- 18 mg/dl before exercise, reached a nadir of 78 +/- 17 mg/dl at 35 min, and were 81 +/- 15 mg/dl at the end of exercise. During the control study at rest, in the same 45-min time interval, plasma glucose decreased from 146 +/- 31 to 128 +/- 31 mg/dl. In the exercise study, one patient began exercising while hypoglycemic, and another patient developed asymptomatic hypoglycemia during exercise. In the control study at rest, one patient showed hypoglycemic glucose concentrations. Throughout the exercise study, plasma free-insulin concentrations decreased (from 32 +/- 5 to 20 +/- 4 microU/ml) as a result of the pharmacokinetics of subcutaneously injected insulin.

Platelet resistance to nitrates in obesity and obese NIDDM, and normal platelet sensitivity to both insulin and nitrates in lean NIDDM.

OBJECTIVE: Previous studies in our laboratory showed that the platelet anti-aggregating effect exerted by insulin, mediated by a nitric oxide (NO)-induced increase of guanosine-3',5'-cyclic monophosphate (cGMP), is lost in the insulin-resistant of obesity and obese NIDDM. It is not clear 1) whether the alterations observed in obese NIDDM patients are attributable to the obesity-related insulin resistance or to diabetes per se and 2) whether insulin-resistant states present a normal or a blunted response to NO. This study has been conducted to investigate 1) the platelet sensitivity to insulin in lean NIDDM and 2) the platelet sensitivity to an NO donor, glyceryl trinitrate (GTN), in obesity and in both lean and obese NIDDM. RESEARCH DESIGN AND METHODS: We determined 1) ADP-induced platelet aggregation and platelet cGMP content in platelet-rich plasma (PRP) obtained from 11 lean NIDDM patients, after a 3-min incubation with insulin (0, 240, 480, 960, 1,920 pmol/l) and 2) ADP-induced platelet aggregation and platelet cGMP content in PRP obtained from 9 obese subjects, 11 lean and 8 obese NIDDM patients, and 18 control subjects, after a 3-min incubation with 0, 20, 40, and 100 mumol/l GTN. RESULTS: Insulin dose-dependently decreased platelet aggregation in lean NIDDM patients (P = 0.0001): with 1,920 pmol/l of insulin, ADP ED50 was 141.5 +/- 6.4% of basal values (P = 0.0001). Furthermore, insulin increased platelet cGMP (P = 0.0001) from 7.5 +/- 0.2 to 21.1 +/- 3.7 pmol/10(9) platelets. These results were similar to those previously described in healthy subjects. GTN reduced platelet aggregation in all the groups (P = 0.0001) at all the concentrations tested (P = 0.0001), but GTN IC50 values were much higher in insulin-resistant patients: 36.3 +/- 5.0 mumol/l in healthy control subjects, 26.0 +/- 6.0 mumol/l in lean NIDDM patients (NS vs. control subjects), 123.6 +/- 24.0 mumol/l in obese subjects (P = 0.0001 vs. control subjects), and 110.1 +/- 19.2 mumol/l in obese NIDDM patients (P = 0.0001 vs. control subjects). GTN dose-dependently increased platelet cGMP in all the groups (P = 0.0001 in control subjects, lean NIDDM patients, and obese subjects; P = 0.04 in obese NIDDM patients). Values reached by obese subjects and obese NIDDM patients, however, were lower than those reached by control subjects (with 100 mumol/l of GTN, P = 0.001 and P = 0.0001, respectively). In healthy control subjects and in obese subjects, the insulin:glucose ratio, used as an indirect measure of insulin sensitivity, was positively correlated to GTN IC50 (r = 0.530, P = 0.008), further suggesting that the sensitivity to NO is reduced in the presence of insulin resistance. CONCLUSIONS: The insulin anti-aggregating effect is preserved in lean NIDDM; platelet sensitivity to GTN in preserved in lean NIDDM but is reduced in the insulin-resistant states of obesity and obese NIDDM. Resistance to nitrates, therefore, could be considered another feature of the insulin-resistance syndrome.

Moderate exercise increases platelet function in type I diabetic patients without severe angiopathy and in good control.

OBJECTIVE: The aim of this study was to investigate whether a 45-min moderate exercise, performed postprandially with a timing that partially prevented the risk of hypoglycemia, was able to modify platelet function in patients affected by insulin-dependent (type I) diabetes mellitus without severe late complications and in a good metabolic control. RESEARCH DESIGN AND METHODS: We submitted 6 male type I diabetic patients (27.2 +/- 3.4 yr; body mass index, 21.4 +/- 0.6 kg/m2; HbA1c, 7.6 +/- 0.9%) on a daily three-insulin injection regimen, without severe late complications of diabetes, to a 45-min moderate exercise (about 50% of maximal oxygen consumption) with a cycle ergometer, beginning 180 min after breakfast and 195 min after a subcutaneous shot of regular insulin. Serial venous blood samples were conducted to measure plasma glucose, free insulin, counterregulatory hormones (glucagon, growth hormone, cortisol, and catecholamines), platelet sensitivity to ADP, platelet activating factor and collagen, and plasma concentrations of the platelet-specific protein beta-thromboglobulin (a marker of the platelet release reaction in vivo). RESULTS: Exercise was accompanied by a decrease of plasma glucose (from 5.9 +/- 1.2 to 4.6 +/- 1 mmol/L, P = 0.067) and free insulin (from 180 +/- 36 to 114 +/- 30 pmol/L, P = 0.003), and by a significant increase of growth hormone (from 5 +/- 1 to 15 +/- 4 micrograms/L, P = 0.045), cortisol (from 240 +/- 30 to 406 +/- 69 nmol/L, P = 0.018), epinephrine (from 1005 +/- 240 to 5143 +/- 1753 pmol/L, P = 0.077), and norepinephrine (from 5.04 +/- 1.08 to 13.48 +/- 2.98 nmol/L, P = 0.009). Platelet sensitivity to the agonists and plasma concentrations of beta-thromboglobulin increased during the exercise period. In particular, ADP ED50 reached during exercise 61 +/- 16% of basal values (P = 0.048), platelet activating factor ED50 reached 73 +/- 11% (P = 0.043), and collagen ED50 reached 68 +/- 9% (P = 0.008). beta-Thromboglobulin rose from 24 +/- 2 to 32 +/- 3 micrograms/L (P = 0.007). CONCLUSIONS: Moderate exercise enhances platelet function in type I diabetic patients without severe angiopathy and in a good metabolic control.

Preliminary results of a phase I/II study of paclitaxel, cisplatin, and cyclophosphamide in advanced ovarian carcinoma.

The clinical activity and toxicity of the triple combination of paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ), cyclophosphamide, and cisplatin was assessed in both previously treated and untreated women with advanced ovarian carcinoma. Paclitaxel 175 mg/m2 was administered over 3 hours following standard premedication (prednisolone, dexchlorpheniramine, and cimetidine). Cisplatin 80 mg/m2 and cyclophosphamide 600 mg/m2 were given 6 to 12 hours after paclitaxel. Treatment was given at 3-week intervals for six cycles. Twenty-seven patients entered the study; 23 were evaluable for toxicity and 17 for response. Paclitaxel appeared to add additional efficacy to the standard cisplatin/cyclophosphamide regimen. Both the overall and complete remission rates were very high (88% and 70%, respectively), and histologically confirmed complete remissions exceeded 60%. Longer follow-up is needed to determine the duration of these responses. The primary toxicities included leukoneutropenia, peripheral neuropathy, asthenia, and alopecia. Only two of 23 patients withdrew because of toxicity, however, and only two treatment cycles were complicated by neutropenic fever requiring intravenous antibiotics. No life-threatening toxicities were encountered, although the peripheral neuropathy was poorly and slowly reversible and may have a significant impact on the patients' quality of life.