Inhibition of platelet aggregation by transdermal glyceryl trinitrate.

OBJECTIVE--To determine the optimum conditions for the demonstration of an antiplatelet effect of nitric oxide and to use these conditions to elucidate the effects of a transdermal glyceryl trinitrate patch on platelet aggregation in normal volunteers. METHODS--An open prospective crossover study. The effects of nitric oxide on platelet aggregation in whole blood and platelet rich plasma as stimulated by adenosine diphosphate and U46619 was assessed in the presence and absence of iloprost and MB22948. Optimum conditions for the demonstration of an antiplatelet effect of nitric oxide were then applied to whole blood from normal volunteers in the presence and absence of a transdermal glyceryl trinitrate patch. SETTING--University hospital. SUBJECTS--Eight normal volunteers. MAIN OUTCOME MEASURES--Platelet aggregation in the presence and absence of transdermal glyceryl trinitrate. RESULTS--The optimum conditions for the demonstration of an antiplatelet effect of nitric oxide in whole blood were collecting blood into a tube containing MB22948 and citrate and stimulating platelet aggregation with adenosine diphosphate in the presence or absence of iloprost. Using this method a significant effect of transdermal glyceryl trinitrate on platelet aggregation was shown (P < 0.03) in the presence and absence of iloprost. CONCLUSIONS--These results are consistent with an inhibitory effect on platelet aggregation of nitric oxide liberated by transdermal glyceryl trinitrate. Optimum test conditions are needed to show this effect. The clinical significance of the antiplatelet effect of transdermal glyceryl trinitrate is unknown.     

Prothrombin complex proteins as cofactors in platelet aggregation. I. Inhibition of aggregation by antiserum

Data are presented to support the concept that the vitamin K-dependent coagulation factors are adsorbed onto the platelet membrane and that these make up part of the "plasma atmosphere" necessary for the aggregation of platelets by various agents. Together with evidence that other coagulation factors also are part of the plasma atmosphere, it is suggested that the aggregation reaction is part of the coagulation sequence. The immunologic approach to demonstrating constituents of the platelet membrane promises to be a highly specific technique for studying further the constituents of the platelet membrane and their reactions in hemostasis.     

Inhibition of platelet aggregation by a monoclonal antibody against human fibronectin.

A monoclonal antibody (A3.3) has been generated against human platelet fibronectin (FN). A3.3 reacts with human plasma FN but with no other plasma proteins. A3.3 was found to inhibit thrombin- or ionophore A23187-stimulated aggregation of gel-filtered platelets in a concentration-dependent manner in both an aggregometer assay and a sensitive well plate aggregation assay. The antibody does not block secretion of serotonin. Four other anti-FN monoclonal antibodies that recognize different epitopes on FN than A3.3 does have no effect on platelet aggregation. A3.3 does not block the adhesion of CHO cells to FN-coated surfaces, indicating that it does not bind to the identified cell-binding domain of FN. A3.3 reacts with a 160/140-kDa doublet, known to contain the cell-binding domain, that is produced by digestion of FN with elastase or thermolysin. However, the antibody does not react with lower molecular weight species that also contain the cell-binding domain or with any of the other identified domains of FN. The A3.3 epitope is extremely protease sensitive and the smallest fragment found in any digest that retains reactivity with A3.3 is a 70-kDa peptide produced in low yield by mild thermolytic cleavage of FN. These data suggest that A3.3 defines a functional site present on both the platelet and plasma FN molecule that has a direct role in platelet aggregation.     

Inhibition of platelet aggregation with histamine

A sensitive in vitro technique was used to demonstrate the inhibitory effects of histamine or human blood platelets. Platelet aggregation by epinephrine was completely inhibited at 10(-3) M concentrations of histamine. Persistent elevations of cyclic AMP levels were shown to occur in the platelets when histamine was added and corresponded to the inhibitory effects on platelet aggregation. In contradistinction to the analogous inhibitory effects by histamine on other cellular elements of the hematopoietic system, this inhibitory effect on platelets could not be blocked by equimolar concentrations of either of the classes of histamine antagonists presently available. It is suggested that there may be other additional histamine receptors on the surface of membranes of platelets or that histamine mediated its inhibitory effect on platelets through a mechanism other than surface receptors.     

Cyclooxygenase-2 inhibitors enhance shear stress-induced platelet aggregation.

OBJECTIVES: We aimed to investigate the effect of parecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, on in vivo shear stress-induced platelet aggregation in a rat model of arterial bypass with focal narrowing. BACKGROUND: Long-term use of COX-2 inhibitors is associated with increased incidence of adverse cardiovascular events, especially in patients with a history of cardiovascular disease. These patients are at risk for thrombotic occlusion of arterial stenoses initiated by shear stress-induced platelet aggregation. METHODS: To mimic the combination of a tight arterial stenosis and high shear stress in rats, an extracorporeal shunt from carotid to femoral artery was compressed by the rollers of a pump. Platelet aggregation was continuously measured by a photometric detector in the shunt. RESULTS: Pretreatment with parecoxib (20 mg/kg) almost doubled shear stress-induced platelet aggregation (188% vs. 100% in control subjects, p = 0.0003). This was accompanied by a fall in plasma 6-keto-prostaglandin F(1alpha) from 100 +/- 25 pg/ml to 36 +/- 11 pg/ml (p < 0.0001). Enhanced platelet aggregation was also observed with high-dose aspirin (150 mg/kg) (146%; p = 0.02) but not with low-dose aspirin (25 mg/kg), which reduced aggregation (68%; p = 0.01). The effect of parecoxib was neutralized by low-dose (1 mg/kg) clopidogrel (from 188% to 92%; p = 0.0001), but not by low-dose aspirin (from 188% to 177%; p = NS). CONCLUSIONS: In the presence of an arterial stenosis, COX-2 inhibitors enhance shear stress-induced platelet aggregation. This enhancement was prevented by low-dose clopidogrel but not by low-dose aspirin. Clopidogrel might therefore allow COX-2 inhibitors to be used without raising risk of thrombotic occlusion.     

Serine protease inhibitors suppress pancreatic endogenous proteases and modulate bacterial neutral proteases

Pefabloc, Trasylol and Urinary Trypsin Inhibitor (UTI) have been reported to be effective serine protease inhibitors that impair pancreatic endogenous proteases resulting in improved islet yield. Here we evaluated the effect of these inhibitors on endogenous proteases (trypsin, chymotrypsin and elastase), bacterial neutral proteases (thermolysin and neutral protease) and islet isolation digestion samples. Protease activity was measured using a fluorimetric assay and islet function was assessed by dynamic perifusion. Trypsin, chymotrypsin and elastase were significantly inhibited by Pefabloc and UTI. Trasylol showed strong inhibitory effects on trypsin and chymotrypsin but also decreased thermolysin activity. UTI was found to inhibit the activity of endogenous proteases and increase the activity of bacterial neutral proteases. Human islets exposed to Pefabloc had reduced insulin response, unlike Trasylol or UTI, which had no detrimental effect on insulin secretion. Although Trasylol was an effective inhibitor of endogenous proteases, FDA regulatory issues preclude its use in clinical application and thus in the isolation process. UTI has the greatest potential because it impairs endogenous pancreatic proteases and enhances digestion enzymes.     

Serine protease inhibitors suppress pancreatic endogenous proteases and modulate bacterial neutral proteases

Pefabloc, Trasylol and Urinary Trypsin Inhibitor (UTI) have been reported to be effective serine protease inhibitors that impair pancreatic endogenous proteases resulting in improved islet yield. Here we evaluated the effect of these inhibitors on endogenous proteases (trypsin, chymotrypsin and elastase), bacterial neutral proteases (thermolysin and neutral protease) and islet isolation digestion samples. Protease activity was measured using a fluorimetric assay and islet function was assessed by dynamic perifusion. Trypsin, chymotrypsin and elastase were significantly inhibited by Pefabloc and UTI. Trasylol showed strong inhibitory effects on trypsin and chymotrypsin but also decreased thermolysin activity. UTI was found to inhibit the activity of endogenous proteases and increase the activity of bacterial neutral proteases. Human islets exposed to Pefabloc had reduced insulin response, unlike Trasylol or UTI, which had no detrimental effect on insulin secretion. Although Trasylol was an effective inhibitor of endogenous proteases, FDA regulatory issues preclude its use in clinical application and thus in the isolation process. UTI has the greatest potential because it impairs endogenous pancreatic proteases and enhances digestion enzymes.     

Inhibition of platelet aggregation by myeloid leukaemic cells demonstrated in vitro

The effect of myeloid leukaemic cells, cells of the promyelocytic cell line HL-60 and normal polymorphonuclear granulocytes (PMN), enclosed in dialysis tubes, on the aggregation of allogeneic normal platelets after induction with arachidonic acid (AA) and with adenosine diphosphate (ADP) was investigated in vitro. Inhibition of aggregation was seen after preincubation of the platelets with leukaemic blood or bone marrow cells from 7/14 patients belonging to various FAB groups and with HL-60 cells, but not with normal PMN (14/14 cases). A dose-dependent inhibition was seen after lysis of some leukaemic cells with anti-human rabbit antiserum and active complement. Lysis of normal PMN inhibited platelet aggregation slightly and inconstantly and only at higher cell concentrations. Platelet inhibitory activity was not related to a higher rate of metabolism of the leukaemic cells which inhibited platelet aggregation since they did not differ from the cells not inhibiting aggregation with respect to heat production. Neither was a non-specifically increased cell membrane permeability the cause of the release of platelet inhibitory factor(s) since the release of 51Cr-labelled leukaemic cells was not related to the inhibition of platelet aggregation.     

Inhibition of cyclooxygenase-independent platelet aggregation by low vitamin E concentration

Platelet aggregation induced by threshold concentrations of agonists such as collagen, PAF or epinephrine was inhibited in vitro by 100 microM aspirin but was restored by stimulating platelets with high concentrations of collagen, PAF or by a combination of epinephrine and PAF. Incubating aspirin-treated platelets with 50-100 microM vitamin E or vitamin E acetate inhibited platelet aggregation by high concentrations of collagen and PAF and by the combination of epinephrine and PAF; platelet thromboxane A2 formation was less than 10% in samples incubated with 100 microM aspirin. Apyrase, added to aspirin-treated platelet, did not influence platelet aggregation induced by epinephrine and PAF. The present study suggests that concentrations of vitamin E as low as 50-100 microM inhibit cyclooxygenase-independent platelet aggregation when combined with an inhibitor of the arachidonate pathway.     

Phorbol-myristate-acetate-induced platelet aggregation in the presence of inhibitors

4 beta-Phorbol-12-myristate-13-acetate (PMA) at 100 ng/ml was able to induce platelet aggregation in the presence of agents which inhibited aggregation, triggered by other agonists such as adenosine diphosphate sodium salt (ADP), thrombin and collagen. PMA induced aggregation in acid-citrate-dextrose platelet-rich plasma. 100 microM tetracaine, 5 microM bromophenacyl bromide and 0.2 mM mepacrine decreased PMA-induced aggregation by only 10% in contrast to their high inhibitory effect on other aggregation systems. However, 0.4 mM mepacrine did inhibit PMA-induced aggregation at the same rate as the other aggregation systems. 100 mg/ml vincristine slightly affected PMA-induced platelet aggregation, whereas cytochalasin B rather enhanced it. Nordihydroguaiaretic acid, 5, 8, 11, 14-eicosatetraynoic acid and p-nitrophenyl-phosphorylcholine had no effect on PMA- or collagen-induced platelet aggregation, partially inhibited aggregation triggered by ADP and strongly inhibited aggregation caused by thrombin. It is suggested that PMA exerts its effect on platelets mainly due to its ability to alter their membranes.     

Collagen-stimulated human platelet aggregation is mediated by endogenous calcium-activated neutral protease

To clarify the physiological role of calcium-activated neutral protease (CANP) in human platelets, we loaded the platelets with a Ca2+ -sensitive fluorescent dye, fura-2, and measured the degree of aggregation, cytosolic calcium ion concentration [( Ca2+]i), and proteolysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). At physiological concentration of Ca2+ (1 mM) in the incubation medium, [Ca2+]i was below 0.5 microM and platelet aggregation was not shown. Ionomycin (0.15 microM) or collagen (50 micrograms/ml), but not ADP (10 microM), sharply enhanced the [Ca2+]i to near 1 microM and caused the aggregation. A calcium entry blocker, verapamil, completely abolished both the [Ca2+]i rise and the aggregation. NCO-700, a membrane permeable inhibitor against cysteine proteases (including CANP), dose-dependently blocked the aggregation but did not change the [Ca2+]i transient. SDS-PAGE revealed that filamin, talin, and 70 kDa protein were specifically degraded when platelets were aggregated by ionomycin or collagen and that the proteolysis was not observed when the aggregation was blocked by verapamil or NCO-700. These data provided evidence that Ca2+ entry exceeding 0.5 microM is essential, but not sufficient per se, and that activation of cysteine protease, most likely CANP, is involved in the platelet aggregation by collagen or calcium ionophore.     

Possible involvement of oxidative stress in exercise-mediated platelet activation

In this study, we have attempted to verify whether a single bout of strenuous exercise performed by sedentary healthy individuals may interfere with the mechanisms controlling platelet sensitivity through exercise-related modifications of plasma oxidant/antioxidant equilibrium. Strenuous exercise resulted in an increased ADP- and collagen-evoked platelet aggregation associated with modified membrane fluidity and ion homeostasis. We also observed an enhanced plasma accumulation of secondary products of lipid peroxidation together with an increased susceptibility of low density lipoprotein (LDL) to in vitro oxidation and a decreased total plasma antioxidant potential. Notably, an acute elevation of nitrite/nitrate (NOx) amount was detected in plasma, whilst a decreased NOx content was measured in platelets. Findings of the current study suggest that oxidative stress induced by acute strenuous exertion may interfere with platelet responsiveness by promoting ox-LDL-mediated platelet activation and by decreasing platelet-derived nitric oxide bioactivity.     

Inhibition of platelet aggregation by aspirin progressively decreases in long-term treated patients

OBJECTIVES: We sought to investigate, during a two-year follow-up period, the effects of aspirin on platelet aggregation. BACKGROUND: The platelets of patients given aspirin may be less sensitive to antiplatelet treatment, although the extent of such phenomenon over long-term follow-up is unclear. METHODS: Adenosine diphosphate (ADP) and collagen-induced platelet aggregation was periodically monitored before and after 2, 6, 12, and 24 months of treatment with aspirin (n = 150) or ticlopidine (n = 80) in patients matched for gender, age, and risk factors for atherothrombosis. RESULTS: Compared with baseline values, two months of aspirin treatment significantly inhibited platelet aggregation; thereafter, this inhibitory effect progressively decreased. At 24-month follow-up, collagen-induced platelet aggregation was significantly higher than that observed at two months (p < 0.05); a more pronounced difference was observed when collagen-induced lag phase was considered (p < 0.01). Restoration of platelet aggregation was less evident when ADP was used as an agonist. Conversely, the inhibition induced by ticlopidine was constant throughout follow-up with both agonists. CONCLUSIONS: The study demonstrates that a long-term treatment with aspirin is associated with a progressive reduction in platelet sensitivity to this drug.     

Lebetin peptides: potent platelet aggregation inhibitors

Lebetins from Macrovipera lebetina snake venom constitute a new class of inhibitors of platelet aggregation. There are two groups of peptides: lebetin 1 (L1; 11- to 13-mer) and lebetin 2 (L2; 37- to 38-mer). The short lebetins are identical to the N-terminal segments of the longer ones. They inhibit platelet aggregation induced by various agonists (e.g. thrombin, PAF-acether or collagen). The shortest lebetin (11-mer) shows potent inhibition of rabbit (IC(50) = 7 nM) and human (IC(50) = 5 nM) platelets. They prevent collagen-induced thrombocytopenia in rats. N- and C-terminal-truncated synthetic L1gamma (sL1gamma; 11-mer) is less active in inhibiting platelet aggregation than the native peptide. Results from Ala scan studies of the sL1gamma peptide indicated that replacement of the residues (P3, G7, P8, P9 or N10) resulted in a remarkable drop in the activity, whereas replacement of residues K2, P4 or K6 by Ala resulted in enhancement of the antiplatelet activity by at least 10-fold. To examine the activity of multimeric L1gamma, several multimeric peptides were synthesized using the multiple-antigen peptide system assembled on a branched lysine core and their antiplatelet activity was evaluated in vitro. The largest multimeric peptides showed a 1,000-fold increase in antiplatelet activity.