Estriol-induced fibrinolysis due to the activation of plasminogen to plasmin by nitric oxide synthesis in platelets.

Estriol, an oestrogen, at 0.6 nmol/l was reported to inhibit ADP-induced platelet aggregation through nitric oxide synthesis. As nitric oxide has been reported to cause fibrinolysis due to the activation of plasminogen to plasmin, the role of estriol as a fibrinolytic agent was investigated. Also, the mechanism of estriol-induced nitric oxide synthesis in anucleated platelets was investigated. The estriol-induced lysis of platelet-rich plasma (PRP) clot was determined by photography of the clot lysis and by the assay of fibrin degradation products in the lysate and was obtained by SDS-PAGE. Nitric oxide was determined by methemoglobin method. The platelet membrane protein was isolated from the platelets by using Triton X-100 (0.05% v/v). The binding of estriol to the protein was determined by Scatchard plot by using an ELISA for estriol. Estriol at 0.6 nmol/l was found to lyse the clotted PRP due to fibrinolysis that produced fibrin degradation products in the lysate. The amino acid analysis of the platelet membrane protein, which resembles with nitric oxide synthase (NOS) activity, was activated nearly 10-fold over the control in the presence of estriol and was identified to be a human serum albumin precursor (Mr. 69 kDa) that binds to estriol with Kd1 of 6.0 × 10 mol/l and 39 ±â€Š2 molecules of estriol bound the NOS molecule. The estriol-induced nitric oxide is capable of inducing fibrinolysis of the clotted PRP. The binding of estriol to platelet membrane NOS activated the enzyme in the absence of DNA in the platelet.

Lower-extremity functional electrical stimulation decreases platelet aggregation and blood coagulation in persons with chronic spinal cord injury: a pilot study.

BACKGROUND: Individuals with spinal cord injury (SCI) develop premature cardiovascular disease. Regular exercise reduces the incidence and symptoms of cardiovascular disease in able-bodied individuals; these salutary effects of exercise have not been documented in persons with SCI. OBJECTIVE: To evaluate the effects of functional electrical stimulation leg cycle ergometry (FES-LCE) exercise training on platelet aggregation and blood coagulation in persons with SCI. PARTICIPANTS: Subjects (n=14) with stable chronic (>1 year) paraplegia (T1-T10) or tetraplegia (C4-C8). METHODS: Blood samples were collected before and after the first and eighth sessions (2 sessions per week for 4 weeks) of FES exercise. RESULTS: Platelet aggregation was inhibited by 20% after the first session and by 40% (P < 0.001) after the eighth session. Thrombin activity was unchanged after the first session (10.7 +/- 0.85 s to 10.43 +/- 0.56 s) and decreased after the eighth session (12.5 +/- 1.98 s to 11.1 +/- 1.7 s; P < 0.0003). Antithrombin III activity increased after the first (103.8% +/- 8.9% to 110% +/- 6.9%; P < 0.0008) and eighth sessions (107.8% +/- 12.1% to 120.4% +/- 13.1%; P < 0.0001). Cyclic adenosine monophosphate increased after the first (9.9% + 2.5% to 15.8% +/- 3%; P < 0.001) and eighth sessions (17.8% +/- 4.2% to 36.5% +/- 7.6%; P < 0.0001). After the eighth session, factors V and X increased significantly (88% +/- 27% to 103% +/- 23%, P < 0.0001; 100% +/- 40% to 105% +/- 7%, P < 0.01, respectively); factors VII and VIII and fibrinogen did not change significantly. A significant reduction in platelet activation/aggregation was demonstrated in response to FES-LCE. The decrease in thrombin level was caused by the simultaneous increase in antithrombin activity. CONCLUSION: These findings provide new insight into the potential protective effects of FES-LCE against the risk of cardiovascular disease.

The role of nitric oxide in aspirin induced thrombolysis in vitro and the purification of aspirin activated nitric oxide synthase from human blood platelets.

Aspirin, a well-known inhibitor of platelet aggregation, is extensively used for the prevention/treatment of coronary artery disease. The beneficial and antithrombotic effects of the compound are related to the inhibition of platelet cyclooxygenase. It is currently believed that aspirin has no effect on the formed thrombus, which results in coronary artery disease. It was found that the exposure of platelets to 4.0 microM aspirin either in vitro or in vivo resulted in fibrinolysis of the formed "clot" produced by the recalcification of platelet-rich plasma due to the production of NO in these cells by the compound. The lysis of clot in the presence of aspirin was found to be related to the fibrinolysis with simultaneous appearance of fibrin degradation products due to the generation of serine proteinase activity by NO in the assay mixture. The aspirin activated nitric oxide synthase that catalyzed the synthesis of NO in platelets was solubilized by Triton X-100 treatment and purified to homogeneity by chromatography on DEAE cellulose and Sephadex G-50 columns. The enzyme was found to be a single chain polypeptide with M.W. 19 kDa. The treatment of human plasminogen with NO was found to directly activate the zymogen to plasmin with the production of preactivation peptide in the absence of cofactors, or cells without the formation of cyclic GMP in the assay mixture.

The thrombolytic effect of aspirin in animal model.

BACKGROUND: The aspirin induced platelet aggregation has been reported to be mediated through the inhibition of platelet prostaglandin synthesis. This compound has also been recently reported to stimulate nitric oxide synthesis in platelets. Since nitric oxide has been reported to produce fibrinogen/fibrinolytic effect, investigation was carried out to determine fibrinolytic effect of in vivo exposure of platelets to aspirin in normal volunteers on the fibrinolysis of the clotted platelet-rich plasma in vitro. The thrombolytic effect of aspirin in situ was also carried out by injecting aspirin solution in the mice with ADP induced formed thrombi in the coronary artery. METHODS AND RESULTS: It was found that the clotted platelet-rich plasma prepared from the volunteers (n = 10, F = 5, M = 5) who ingested 150 mg aspirin, began to undergo spontaneous and progressive fibrinolysis for 200 min at 37 degrees C with the generation of fibrin degradation products in the lysate. No such fibrinolysis could be seen in control experiments. When platelet thrombi were produced in the coronary artery of mice by injecting ADP, and these animals subsequently received intravenous injection of aspirin (4 muM final), they not only survived (P < 0.0001, n = 10) the thrombogenic assault but the lysis of the platelet thrombi was also noted in the post mortem examination. The thrombolytic effect of aspirin was found to be comparable to that of streptokinase in these animals. CONCLUSIONS: Aspirin, through the stimulation of NO synthesis, may produce thrombolysis in vivo.

Restoration by aspirin of impaired plasma maspin level in human breast cancer.

Maspin, an anti breast cancer protein, is produced in the normal mammary cells but not in malignant cells in breast cancer. We investigated the effect of aspirin induced increase of plasma nitric oxide (NO) on plasma maspin production in breast cancer patients. Fifteen breast cancer patients (35-65 years), who had not yet undergone any cancer therapy, and an equal number of age matched normal female volunteers participated in the study. They were asked not to take any medication for two weeks. All participants then ingested 150 mg of aspirin. Plasma NO and maspin levels were determined before and at 60 min after the ingestion of aspirin. It was found that the maspin level in plasma increased to 4.63+/-0.02 nM from the basal 0.95+/-0.012 nM (p<0.001) with increase of plasma NO from 0.60+/-0.03 microM to 2.08+/-0.030 microM (p<0.001) in breast cancer patients. In normal volunteers the basal maspin increased from 4.76+/-0.041 to 9.36+/-0.036 nM (p<0.001) with increase of NO from 2.15+/-0.08 to 3.36+/-0.04 microM (p<0.001) at the same period. These results indicated that the ingestion of aspirin might be beneficial for breast cancer through increased maspin production.

Effects of oxandrolone, an anabolic steroid, on hemostasis.

This study evaluated the short-term effects of oxandrolone, an anabolic androgenic synthetic steroid, on blood coagulation and the hemostatic/fibrinolytic system in healthy individuals. Subjects (n = 14) were administered oxandrolone (10 mg twice daily) for 14 days. Blood was obtained on days 0, 1, 3, 7, 9, 14, and then at day 42 (28 days after discontinuation of the drug). Samples were analyzed for the plasma plasminogen, plasminogen activator inhibitor (PAI-1), fibrinogen, and coagulation factors (II, V, VII, VIII, and X). After 7 days of administration of oxandrolone, the plasma plasminogen level significantly increased [100% +/- 21% to 174% +/- 21% (P < 0.0001)]. PAI-1 was significantly decreased at day 3 [16 +/- 9 to 7 +/- 4 mg/dL (P < 0.01)]. Coagulation factors II and V significantly increased at day 14 [88 +/- 15 to 122 +/- 11 (P < 0.005) and 105 +/- 21 to 179 +/- 36% (P < 0.0001)], respectively. Factor VII level decreased by day 3 [91% +/- 26% to 83% +/- 18%, NS], but after 14 days factor VII level returned to baseline (91% +/- 26% to 93% +/- 19%, NS). The increase of factor VIII level was not significant (111% +/- 64% to 125% +/- 55%, NS). Factor X increased steadily over 14 days of drug treatment [96% +/- 11% to 107% +/- 25%, NS] and after discontinuation, decreased and returned to baseline by day 42 [107% +/- 25% to 89% +/- 25%, NS]. Fibrinogen decreased by 22% +/- 12%, (NS). Administration of oxandrolone, to healthy young men was associated with a significant increase in select blood coagulation factors and plasminogen. These changes create a state of potential hypercoagulability that appears to be counterbalanced by increased fibrinolytic activity to maintain homeostasis.

Appearance of a novel prostacyclin receptor antibody and duration of spinal cord injury.

BACKGROUND: Cardiovascular disease (CVD) appears to be accelerated in individuals with chronic spinal cord injury (SCI). Previously, we have identified a novel circulating antibody (IgG) in persons with SCI that specifically blocks the high-affinity prostacyclin (PGI2) receptors on the platelet surface without affecting the low-affinity PGI2 receptors. OBJECTIVE: In this study, the relationship between the time course after SCI to the development of IgG to the high-affinity PGI2 receptor was determined. METHODS: Blood samples were collected 1, 3, 5, 10, and > 10 (15 +/- 4) years after SCI (n = 36). Plasma samples (50 microg) were analyzed by polyacrylamide gel electrophoresis (PAGE) followed by densitometry. RESULTS: The optical density (OD) of the IgG (molecular weight 47,000) at 1 year after SCI was significantly higher than control (1.65 +/- 0.08 vs 1.33 +/- 0.04; P < 0.01). This anti-receptor IgG appears to increase for 5 years and then plateau. At 5 years, 6-10 years, and > 10 years of injury, the OD was 1.83 +/- 0.09, 1.83 +/- 0.10, and 1.87 +/- 0.08, respectively. With an increase in this specific IgG, there was a concomitant decrease in the binding of prostacyclin to its high-affinity receptors on SCI platelets, (non-SCI vs 1, 3, and 5 years after injury; n1 = 172 +/- 25 vs 153 +/- 15, 107 +/- 25, and 40 +/- 4 sites/platelet, respectively; P < 0.001), with no significant change in receptor affinity. CONCLUSIONS: The level of the high-affinity PGI2-receptor antibody determined in individuals with SCI was associated with the duration and not with the level of injury. Platelets from subjects with SCI had a reduction in numbers of high-affinity receptors.

Circulating heavy chain IgG, a pathological mediator for coronary artery disease, recognizes platelet surface receptors of both prostacyclin and insulin.

Although an increased incidence of premature cardiovascular disease has been determined to be the major cause of mortality in subjects with chronic spinal cord injury the identity of the pathophysiological mediators of cardiovascular disease in spinal cord injury remains obscure. Because both insulin and prostacyclin could be important in the prevention of thrombosis, the status of insulin-induced nitric oxide production and the prostacyclin high-affinity receptor interaction in platelets in subjects with spinal cord injury was studied. It was established that the insulin-induced nitric oxide synthesis in platelets from spinal cord-injured subjects was markedly impaired (0.053-0.058, P = 0.37-0.44) compared to (0.062-0.53 microM/10(8) platelets, P < 0.001) due to the presence of a free heavy chain IgG (Mr 47 kDa) in the circulation of subjects with spinal cord injury. This IgG not only blocked insulin receptor binding sites (without affecting dissociation constant of the hormone binding, Kd1 = 2 x 10(-9) M) for the synthesis of nitric oxide but also blocked the prostacyclin receptor interaction in normal platelets. Since the presence of circulating heavy chain of IgG could block the antithrombotic effect of both insulin and prostacyclin, the free heavy chain of the IgG molecule was thought to be one of the pathological mediators for the increased incidence of cardiovascular disease in individual with spinal cord injury. The cross-reactivity of the free heavy chain with two different receptors antigens was thought to be related to the presence of several regions of homology in the amino acid sequence in the insulin and prostacyclin receptor molecules.