Stimulated Platelet Aggregation Research Articles

Species differences in platelet responses to thrombin and SFLLRN. Receptor-mediated calcium mobilization and aggregation, and regulation by protein kinases

The thrombin receptor on human platelets is activated by thrombin to stimulate platelet aggregation through the tethered ligand SFLLRN. This study examined the effects of thrombin and SFLLRN on aggregation and calcium mobilization ([Ca 2+] i) in rat, guinea pig, rabbit, dog, monkey, and human platelets, and the role of protein kinases in regulating these functions. Thrombin induced platelet aggregation and [Ca 2+] i in all species studied; however, only guinea pig, monkey and human platelets were responsive to SFLLRN. Similar species specific effects were obtained with [Ca 2+] i studies. The kinetic profile for [Ca 2+] i differed among species, suggesting that regulatory mechanisms for calcium differed between agonists and among species. Staurosporine, a non-selective inhibitor of protein kinases, inhibited platelet aggregation induced by thrombin or SFLLRN in all species. Staurosporine inhibited thrombin-induced [Ca 2+] i in guinea pigs, had no effect in rat, and increased [Ca 2+] i in all other species. Staurosporine inhibited SFLLRN-induced [Ca 2+] i in guinea pig, yet had no effect in monkey or human. Tyrphostin 23, a specific inhibitor of tyrosine protein kinases, inhibited thrombin-induced aggregation of rabbit, monkey, dog and human platelets. SFLLRN-induced aggregation was also inhibited by tyrphostin 23. Tyrphostin 23 inhibited [Ca 2+] i induced by either thrombin or SFLLRN in all species. Based on the differential response to agonist stimulation, we propose that thrombin can activate platelets via SFLLRN-dependent and independent mechanisms, which could involve yet unrecognized subtypes of the thrombin receptor or distinct cellular activating mechanisms. Furthermore, differential regulation of calcium mobilization and aggregation was observed in those platelets responding to either thrombin or SFLLRN.

Accumulation of a Right Ventricular Mass During Shoulder Hemiarthroplasty

Accumulation of a Right Ventricular Mass During Shoulder Hemiarthroplasty

Oxidized LDL and Reduction of the Antiaggregating Activity of Nitric Oxide Derived from Endothelial Cells

Oxidized LDL has been observed to induce abnormalities in endothelial function which may be relevant for the progression of atherosclerotic lesions. We studied in vitro the possible effects of oxidized LDL on the antiaggregating activity of endothelial cells, which is dependent on release of prostacyclin and nitric oxide. We used an experimental model in which cultured human endothelial cells were placed in the aggregometer in contact with human platelets, after blockade of cyclo-oxygenase by adding acetylsalicylic acid. In this way the antiaggregant effect of endothelial cells was dependent on the release of nitric oxide alone; prevention of antiaggregant activity by preincubation of endothelial cells with 300 microM L-NG-mono-methylarginine confirmed this. When this system was used, endothelial cells (2-7.5 x 10(5)/ml) almost completely inhibited thrombin-induced (0.02-0.08 U/ml) platelet aggregation (2 x 10(8) platelets/ml), measured according to Born (11.1% +/- 8.5 vs 68.6% +/- 12.6, M +/- SD). This antiaggregating activity was reduced when slightly oxidized LDL 100 micrograms/ml (35.2% +/- 14.9, p < 0.001), but not native LDL 100 micrograms/ml (7.5% +/- 7.6), was added immediately before aggregation was induced. Incubation of endothelial cells with oxidized LDL 100 micrograms/ml for 1 h did not affect the antiaggregating capacity, unless oxidized LDL was present during aggregation (18.3% +/- 10.2 vs 35.8% +/- 9.6, p < 0.02). No significant direct effect of either oxidized or native LDL on stimulated platelet aggregation was observed. Our results indicate that slightly oxidized LDL can reduce the antiaggregating properties of the endothelium, probably by interaction with NO rather than through inhibition of its synthesis.

Increased production of eicosanoids, TXA2, PGI2 and LTC4 in experimental spinal cord injuries

Arachidonate metabolites have many kinds of bioactivities. Thromboxane A2 (TXA2) stimulates platelet aggregation and vasoconstriction, whereas prostaglandin I2 (PGI2) antagonises its activities. Thromboxane B2 (TXB2) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) are determined in biological materials. Production of TXB2, 6-keto-PGF1 alpha and leukotriene C4 (LTC4), which have potent vascular permeability, was measured by radioimmunoassay in experimental spinal cord injured animals. TXB2 level in the rat spinal cord reached a peak concentration of 133.6 +/- 3.8 pmol/g cord, and 6-keto-PGF1 alpha increased to 26.2 +/- 11.7 pmol/g cord 5 minutes after the injury. There was good correlation between TXB2 production and vascular damage as monitored by fluorescein uptake. When OKY-046 ((E)-3-[4-(1-imidazolylmethyl) phenyl]-2-propenoic acid), which selectively inhibits TXA2 synthetase activity, was administered 10 minutes before injury, the increase in TXB2 production was inhibited by more than 80%, but the degree of vascular damage was reduced by only 40%. In the guinea pig spinal cord, LTC4 levels reached a peak concentration of 2.2 +/- 0.4 pmol/g cord 10 minutes after compression, while that of TXB2 reached 146.8 +/- 6.2 pmol/g cord. The increased production of TXB2 was correlated with the degree of compression injury while that of LTC4 production did not. These findings suggest that vasoactive eicosanoids, TXA2, PGI2 and LTC4, play important roles in secondary damage following spinal cord injury, although their roles may be different among species of animals.

Thrombin-induced platelet aggregation, phosphoinositide metabolism and protein phosphorylation in NIDDM patients treated by diet, sulphonylurea or insulin

We studied thrombin-induced metabolism of phosphoinositide, protein phosphorylation and platelet aggregation in platelets from 32 NIDDM patients and 12 control subjects. To clarify the effect of diet, sulphonylureas, or insulin treatment, the subjects were divided into three groups based on the type of treatment. Thrombin-induced platelet aggregation was measured with an aggregometer. Low-dose thrombin (0.25 U/ml)-stimulated platelet aggregation in diabetic patients was significantly increased compared with the control subjects. Platelet aggregation in the sulphonylurea and insulin groups was significantly lower than in the diet group. On the other hand, in platelets incubated with [32P]orthophosphate, thrombin-induced incorporation of 32P radioactivity into phosphatidic acid (PA) was significantly lower in the sulphonylurea and insulin groups than in the diet group. Thrombin-induced incorporation of [32P] radioactivity into phosphatidylinositol (PIP) for 10 s was significantly higher in the sulphonylurea group than in the diet group. There were no differences in thrombin-induced 47 kDa protein phosphorylation between platelets from the diet, sulphonylurea, or insulin groups. These results suggest that sulphonylureas and insulin induce suppression of thrombin-induced activation of phospholipase C, which mediates hydrolysis of PIP and PIP2 and production of PA, which leads to inhibition of platelet aggregation.

Effect of the antihistaminic drug Bromadryl on ex vivo platelet functions during gestation in rats.

The effect of the antihistaminic drug Bromadryl on stimulated platelet aggregation was studied in pregnant rats. Bromadryl was administered orally in doses 1, 5 and 10 mg/kg from day 2 to day 19 of gestation. On day 20 of gestation blood samples were taken and the aggregation of platelets was measured after stimulation with 9 different concentrations of ADP in the concentration range 0.2 to 100 mumol/l. During gestation, platelet aggregation was significantly increased in comparison to non-pregnant controls. This was indicated in pregnant rats by the increased maximum amplitude of the aggregation curve as well as by the decreased mean effective concentration of ADP. The treatment of dams with Bromadryl during pregnancy resulted in decreased platelet aggregation. The dose-dependent inhibitory effect of the drug was more pronounced at higher stimulus concentrations. The presented results confirmed the antiplatelet activity of Bromadryl observed previously in vitro.

Antihistaminic drug bromadryl and stimulated platelet aggregation

The H1-receptor antagonist bromadryl dose-dependently inhibited stimulated rat platelet aggregation in vitro. Bromadryl was 10 times more effective on the secondary aggregation of thrombin-stimulated platelets compared with its inhibition of ADP-stimulated primary platelet aggregation in plasma. The inhibition of aggregation was accompanied by a dose-dependent inhibition of thrombin-stimulated malondialdehyde formation and thromboxane B2 production. The results indicate that bromadryl may interfere intracellularly with membrane phospholipid peroxidation and the arachidonic acid metabolism of stimulated platelets. Bromadryl, like other cationic amphiphilic drugs, may inhibit stimulated platelet functions by decreasing the stimulus-induced activation of phospholipase A2. Our results support the possible interference of bromadryl with histamine as an intraplatelet messenger responsible for aggregation.

The role of intracellular calcium in A-23187 stimulated and β-adrenoceptor blocking drug treated blood platelets

A significant concentration-dependent difference was found between β-adrenoceptor blocking drugs in their ability to inhibit A23187-induced isolated platelet aggregation. In the absence of extracellular calcium ions the following rank order of potency to inhibit calcium ionophore stimulated platelet aggregation was shown: propranolol > bevantolol > alprenolol > metipranolol > oxprenolol > atenolol > pindolol > metoprolol ≈ sotalol ≈ practolol. The interruption of induced aggregation, as well as inhibition of aggregation, in the absence of extracellular calcium ions indicated interference of inhibitory β-adrenoceptor blocking drugs with intramembrane or intraplatelet calcium pools activated with A23187. This suggestion was supported by the reversal of the inhibitory effect of β-adrenoceptor blocking drugs in the presence of extracellular calcium ions. The effect was dose dependent and occurred within 30 sec after calcium administration. The results indicated that inhibitory β-adrenoceptor blocking drugs, possessing a cationic amphiphilic structure, suppressed calcium mobilization in A23187-stimulated platelets, most probably after entering platelets. This explains why lipophilic drugs are more effective than hydrophilic ones in calcium ionophore A23187-stimulated platelets.

Characterization of platelet activity in neuroblastoma

A study was conducted to characterize the platelet aggregation induced by neuroblastoma tissue to investigate the mechanism of hypercoagulability in patients with neuroblastoma. The patients whose tumor tissues were examined had been shown clinically to have enhanced platelet activity. Platelet aggregation induced by neuroblastoma tissue extract was compared with that of other pediatric tumors. The effects of pretreatment with an antithrombin agent and prostacyclin (PGI 2) on the platelet aggregation induced by tumor tissue extracts were also evaluated. Tissue extracts of 12 of 15 neuroblastomas, 3 of 3 Wilms' tumors, and 1 pheochromocytoma were demonstrated to have an activity that potentiated platelet aggregation in vitro. The platelet aggregation induced by tissue extracts of neuroblastomas and other tumor tissues was suppressed almost completely by pretreatment with a PGI 2 analogue. The aggregation induced by neuroblastomas and the pheochromocytoma was also suppressed by pretreatment with an antithrombin agent, argatroban, whereas the aggregation induced by Wilms' tumors was not suppressed by this agent. These results suggest that (1) malignant tumors in children also have some chemical substances that sensitize platelet activity, such as those in adult cancers, and (2) thrombin is one of the mediators stimulating platelet aggregation in cases of neuroblastoma, although it is unlikely to be a contributing factor in other pediatric malignancies such as Wilms' tumor.

Low dose acetylsalicylic acid in the antithrombotic treatment of patients with stable angina pectoris and acute coronary syndromes (unstable angina pectoris and acute myocardial infarction).

Acetylsalicylic acid has an antithrombotic effect by inhibition of thromboxane A2 synthesis in platelets. Thromboxane A2 is a potent stimulator of platelet aggregation and vasoconstriction and synthesis may be completely inhibited by a single oral dose of 150 mg acetylsalicylic acid or an intravenous dose of 100 mg. A daily maintenance dose of 75 mg acetylsalicylic acid is sufficient to effectively inhibit thromboxane A2 synthesis in long-term treatment. Acetylsalicylic acid therapy reduces acute myocardial infarction and sudden death in patients with stable angina pectoris and the drug is equally effective in patients with symptomatic and 'silent' angina pectoris. Early intervention with acetylsalicylic acid in patients with unstable angina pectoris reduces the risk of acute myocardial infarction and death. In patients with acute myocardial infarction, acute therapy with acetylsalicylic acid significantly reduces mortality both in monotherapy and in combination with thrombolytics. In the secondary prophylaxis following acute myocardial infarction, acetylsalicylic acid reduces the incidence of reinfarction and coronary death. Treatment of 100 patients with acute coronary syndrome (unstable angina pectoris or acute myocardial infarction) for 2 years may hinder the development of 3-4 fatal and 4 non-fatal vascular events. The risk of gastrointestinal side-effects and bleeding during acetylsalicylic acid therapy is dose-dependent and the incidence is low with a daily dose of 75-150 mg.

A simple assay for ecto-5′-nucleotidase using intact pulmonary artery endothelial cells: Effect of endotoxin-induced cell injury

Adenosine may be protective in acute vascular injury by inhibiting platelet aggregation and neutrophil oxidant release. In contrast, adenine nucleotides, which may be released with acute vascular injury, stimulate platelet aggregation and neutrophil oxidant release. Ectonucleotidases, membrane enzymes that catabolize extracellular nucleotides, are the primary mechanism for degrading circulating nucleotides to adenosine. Ecto-5′-nucleotidase converts extracellular AMP to adenosine. We hypothesized that endothelial cell injury alters ecto-5′-nucleotidase activity. Using a novel assay first reported by Jamal et al. ( Biochem J 250: 369–373, 1988) with rat adipocytes, we studied the properties of ecto-5′-nucleotidase in intact monolayers of cultured bovine pulmonary artery endothelial cells (BPAEC) and examined the effect of endotoxin on enzyme activity. The assay uses a fluorescent analog of AMP, 1, N 6-etheno-AMP (E-AMP), as the substrate for ecto-5′-nucleotidase, and measures ethenoadenosine (E-Ado) formation. Etheno-AMP in Hepes buffer, pH7.4, at 22°, was added to confluent monolayers of BPAEC; samples of supernatant were collected after various intervals, and E-AMP and E-Ado were quantitated by HPLC. Using these methods we found a K m of 15 ± 6 μM, a pH optimum of 7.48, minimal effect of MgCl 2 or CaCl 2 at physiologic pH, and inhibition by α,β-methylene ADP, a known 5′-nucleotidase inhibitor. We established that the monolayer assay was indeed measuring cell surface associated 5′-nucleotidase. To determine the effect of endotoxin, we incubated confluent monolayers with endotoxin in Minimal Essential Medium plus 10% fetal bovine serum for 24 hr, washed them, and assessed the conversion of E-AMP to E-Ado by the endotoxin-injured cells. Endotoxin stimulated endothelial ecto-5′-nucleotidase activity. This increase in 5′-nucleotidase activity in response to endotoxin injury may represent an important clearance mechanism for circulating adenine nucleotides and may be protective in acute vascular injury by increasing adenosine production.

Low zinc status in rats impairs calcium uptake and aggregation of platelets stimulated by fluoride.

Platelets from rats of low zinc status exhibit impaired aggregation in response to ADP stimulation. The abnormality has been traced to defective uptake of calcium from the external medium. This study was designed to determine the location of the molecular defect and whether or not the ADP receptor is involved. Washed platelets were collected from rats fed a low zinc diet (< 1 mg/kg) and control groups that consumed a zinc-adequate diet (100 mg/kg), ad libitum- and pair-fed. Fluoride, a G-protein stimulant, was used to bypass the ADP receptor. F- stimulated platelet aggregation and calcium uptake; both of these functions were impaired by zinc deficiency. At 10 mM F-, the time to half maximal aggregation was increased from 1.8 min in platelets from control to 2.8 min in zinc deficient rats. At 8 mM F-, the uptake of calcium was decreased from 170 to 85 nM cytosolic free calcium. At this concentration of F- there was no release of internal calcium. The results show that the molecular defect in the zinc-deficient platelet is located in the aggregation pathway beyond the ADP receptor and suggest a point between, or including, a G-protein and the plasma membrane calcium channel.

Increased phosphatidic acid and decreased lysophosphatidic acid in response to thrombin is associated with inhibition of platelet aggregation.

Thromboxane A2, produced from the arachidonic acid released from platelet phospholipids by phospholipase A2, stimulates platelet aggregation. It remains unresolved whether additional products of platelet phospholipase A2 might promote aggregation. To address this question, we have used aspirin-treated platelets to block thromboxane A2 formation and studied the influence of the phospholipase A2 inhibitor U10029A on platelet aggregation and secretion in response to thrombin. U10029A at 100 microM markedly inhibited platelet aggregation, but had no effect on platelet secretion. Since this concentration of U10029A effectively blocked lysophosphatidic acid (LPA) formation, LPA was added and found to substantially reverse the inhibitory effect of U10029A in these platelets. Furthermore, the action of U10029A was not due to inhibition of phosphatidate phosphohydrolase because U10029A, unlike propranolol, did not inhibit this enzyme. Although it is not possible to conclusively rule out an effect of U10029A in addition to its inhibition of phospholipase A2, our results reveal that a product of phospholipase A2 other than thromboxane A2 is important for platelet aggregation, but not for secretion in response to thrombin. Our data suggest that this product is LPA. Since the amount of phosphatidic acid (PA) increased dramatically concurrent with inhibition of platelet aggregation, it is safe to conclude that PA has no direct role to promote platelet aggregation in response to thrombin.

Lipoprotein glycation and its metabolic consequences.

In people with diabetes, glycation of apolipoproteins correlates with other indices of recent glycemic control, including HbA1. For several reasons, increased glycation of apolipoproteins may play a role in the accelerated development of atherosclerosis in diabetic patients. Recognition of glycated LDL by the classical LDL receptor is impaired, whereas its uptake by human monocyte-macrophages is enhanced. These alterations may contribute to hyperlipidemia and accelerated foam-cell formation, respectively. Glycation of LDL also enhances its capacity to stimulate platelet aggregation. The uptake of VLDL from diabetic patients by human monocyte-macrophages is enhanced. This enhancement may be due, at least in part, to increased glycation of its lipoproteins. Glycation of HDL impairs its recognition by cells and reduces its effectiveness in reverse cholesterol transport. Glycation of apolipoproteins may also generate free radicals, increasing oxidative damage to the apolipoproteins themselves, the lipids in the particle core, and any neighboring macromolecules. This effect may be most significant in extravasated lipoproteins. In these, increased glycation promotes covalent binding to vascular structural proteins, and oxidative reactions may cause direct damage to the vessel wall. Glycoxidation, or browning, of sequestered lipoproteins may further enhance their atherogenicity. Finally, glycated or glycoxidized lipoproteins may be immunogenic, and lipoprotein-immune complexes are potent stimulators of foam-cell formation.

Hypothesis for the receptors of human blood platelet aggregation and its inhibition by structure-activity relationship

We tried to clarify the size and the common charge distribution of the inhibition or stimulation of human platelet aggregation by structure-activity relationship. Numerous inhibiting and stimulating agents were able to enter the receptors. Inhibitory receptor had recess of 14 × 12.5Å in diameter. Stimulatory receptor had recess of 11 × 12Å in diameter. In the recess, there were three charges, two negative and one positive in the inhibitory receptor, and one negative and two positive in the stimulatory receptor, respectively. Charge distributions and conformation of inhibiting or stimulating agents were similar for the inhibitory agents, prostaglandin I 2 (PGI 2), PGD 2, PGE 1 adenosine and isoproterenol and conformation of the stimulating agents, thromboxane A 2 (TXA 2), platelet activating factor (PAF), adenosine diphosphate (ADP) and adrenaline. Each molecule had 3–10 inhibiting and stimulating conformations. The ratio of the number of conformations for inhibition and stimulating of platelet aggregation was highest for PGI 2 which showed the strongest inhibitory activity. TXA 2 was opposite in both respects.

Therapeutic lidocaine concentrations have no effect on blood platelet function and plasma catecholamine levels.

The effects of therapeutic plasma concentrations of lidocaine on blood platelet function and plasma catecholamine levels were assessed in 9 healthy subjects. There were no significant effects on plasma levels of beta-thromboglobulin, collagen and adenosine diphosphate-stimulated platelet aggregation, thromboxane-B2-concentration in plasma after collagen and ADP stimulated platelet aggregation, or on plasma nor-adrenaline and adrenaline. No significant correlation could be demonstrated between any of the variables tested. Thus, it appeared that lidocaine had no effect on platelets that could be of benefit in acute myocardial infarction. It should be possible to use lidocaine, in combination with thrombolytic therapies without increasing the risk of bleeding complications.

Platelet-activating factor synthesis by peritoneal mast cells and its inhibition by two quinoline-based compounds

1. Peritoneal mast cells from rat were co-incubated in vitro in a platelet aggregometer cuvette with washed rabbit platelets. In response to stimulation with calcium ionophore (A23187; 1-5 microM), the mast cells released a substance which stimulated the platelets to aggregate. These concentrations of ionophore did not stimulate platelet aggregation in the absence of mast cells, nor affect the responsiveness of the platelets to aggregation induced by thrombin or PAF. Release of a PAF-like substance was also observed in response to stimulation of the mast cells with antigen. 2. This pro-aggregatory activity is attributable to the release of PAF by the mast cells, since the activity could be abolished by preincubating the platelets with a specific PAF receptor antagonist (WEB 2086; 10 microM). Furthermore, the platelet-aggregating factor co-migrated with PAF on thin-layer chromatographs and could be abolished by incubation with phospholipase A2 (20 micrograms ml-1) or a specific antibody directed against PAF. 3. The release of PAF by peritoneal mast cells could be inhibited, in a concentration-dependent manner, by PF-5901 (IC50 of 3.9 microM) or Wy-50,295 (IC50 of 1.2 microM), two structurally similar compounds with inhibitory effects on leukotriene synthesis, as well as leukotriene D4 (LTD4) receptor antagonist properties. 4. Inhibition of PAF synthesis was not observed when the mast cells were incubated with a structurally unrelated 5-lipoxygenase inhibitor (A-64077), a structurally dissimilar inhibitor of 5-lipoxygenase activating protein (MK-886) or with a structurally related LTD4 receptor antagonist (MK-571) which lacks inhibitory effects on leukotriene synthesis, each at concentrations of up to 100 microM.5. Neither PF-5901 nor Wy-50,295 (1 or 10 microM) significantly affected histamine release or prostaglandin D2 synthesis by peritoneal mast cells in response to calcium ionophore stimulation.6. These results demonstrate the ability of a class of quinoline-based compounds to inhibit PAF synthesis by peritoneal mast cells. This activity does not appear to be related to effects of these compounds on leukotriene synthesis or LTD4 receptors. The ability of these compounds to inhibit PAF synthesis may contribute to their anti-inflammatory properties.

Thrombosis: A common arrival point of different pathways

The cause and cure of thrombosis remain unknown. The majority of arterial thrombotic events occur in patients without obvious risk factors. The sensitivity and specificity of the known risk factors is poor, although several investigators have devised risk calculators in which the cumulative effect of risk factors is summated. Arterial thrombi usually occur in arteries previously affected by atherosclerosis. As staled by Virchow ( 1856) important determinants of thrombosis are the nature of the vessel, the type of flow within it, and the nature of the plasma components. The importance of rupture of the atherosclerotic plaque, causing haemorrhage within the plaque and thrombus formation on it has been emphasised. For instance, there is angioscopic evidence that myocardial infarction is caused by complete occlusive thrombus, whereas unstable angina is secondary to non-occlusive thrombus. The localisation of thrombosis is determined by the anatomical shape of the vessels, being greatest at areas of blood flow disturbance at curves or collaterals in the vessel. Partial thrombotic occlusion leads to further blood flow disturbance, precipitating thrombosis. The clinical importance of the platelet in thrombus formation has come, paradoxically, from the undoubted beneficial effect of aspirin in thrombosis which lowers clinical arterial events by about 20%. Although there is a wealth of biochemical evidence suggesting that platelets may become activated in patients with arterial disease, tests of platelet function to identify individuals al risk of thrombosis have been disappointing. At present they lack the sensitivity and specificity lo provide useful clinical information lo predict the course of arterial disease in a single individual. The plasma components contributing to thrombosis consist of the coagulation and fibrinolytic proteins. Coagulation occurs in a series of complex steps leading to the formation of thrombin, which both clots fibrin and stimulates platelet aggregation. The intrinsic coagulation pathway is triggered by factor XII, whereas the extrinsic pathway is initiated by tissue factor from damaged endolhelial cells or tissues. Finally, the fibrinolytic enzyme is responsible for removal of fibrin in the thrombus. The two main plasminogen activators are tissue type plasminogen activator ((PA) which is derived from intact endolhelium and urokinase type activator (uPA) which is excreted in urine and secreted by kidney and other cells. Their action is promoted by the presence of fibrin and inhibited by plasminogen activators (PAI). The present concept that thrombosis can be promoted by reduction of plasminogen activator activity and increased PAI activity needs further support from clinical studies. In summary, abnormalities in all four major pathways to thrombosis, the vessel wall, the platelets, coagulation and fibrinolysis may all contribute to thrombus formation.

Antiaggregatory Properties of Beta-adrenoceptor Blocking Drugs are Related to their Physico-chemical Properties

The effects of the beta-adrenoceptor blockers (BAB) alprenolol, atenolol, metipranolol, oxprenolol, practolol and propranolol on platelet aggregation stimulated by ADP, thrombin and collagen were investigated. The BAB were divided into three groups according to their liposolubility and antiaggregatory activity. The first group consists of the highly active drugs alprenolol and propranolol; metipranolol and oxprenolol are in the second group; the least active atenolol and practolol are in the third group. Collagen induced platelet aggregation was inhibited by BAB to the highest extent, followed by inhibition of thrombin stimulated aggregation. ADP-induced aggregation was inhibited to the lowest extent. Consistent with the rank order of their partition coefficients, BAB inhibited aggregation with the following order of potency: propranolol > alprenolol > metipranolol > oxprenolol > practolol > atenolol. A positive correlation was found between inhibition of thrombin and collagen stimulated platelet aggregation and the partition coefficients of the individual drugs. A significant time-dependent inhibition of platelet aggregation developed within 30 s of exposure of blood platelets to BAB.

Monoclonal antibodies to ligand-occupied conformers of integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) alter receptor affinity, specificity, and function.

Occupancy of integrin receptors induces conformational changes in the receptor, resulting in exposure of novel interactive sites termed ligand-induced binding sites (LIBS). We report here that Fab fragments of certain antibodies against LIBS on integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa) block platelet aggregation. Thus, certain LIBS or the regions surrounding them may participate in events required for platelet aggregation. In addition, certain anti-alpha IIb beta 3 LIBS Fab fragments stimulated platelet aggregation. This was due to induction of fg binding to alpha IIb beta 3, apparently by shifting a conformational equilibrium between a "resting" and an "activated" state of alpha IIb beta 3. Some of the activating anti-LIBS Fab fragments also induced high affinity fibronectin binding to alpha IIb beta 3, whereas others did not. Thus, changes in the conformation of this integrin modulate both the specificity and affinity of ligand recognition.