Collagen-stimulated Platelet Aggregation Research Articles

Inhibitory effects of cardiotonic pills on platelet function in dogs fed a high-fat diet

Insulin resistance and the consequent metabolic disorders are associated with a state of platelet hyperactivity. Oxidative stress is responsible for the persistent platelet activation. We sought to study the inhibitory effect of cardiotonic pills, an oral herbal component, on platelet function in a dog model with insulin resistance induced by high-fat feeding. We fed 18 dogs with a high-fat diet and six dogs with normal chow as control for 6 months. Then, six dogs were fed with a high-fat diet and received additional aspirin (250 mg/day), and another six dogs received additional cardiotonic pills (1,000 mg/day) for 4 months. Time-course changes in metabolic parameters and platelet function were detected. After high-fat feeding for 6 months, 18 dogs developed a series of metabolic disorders including obesity, dyslipidemia, oxidative stress and insulin resistance. In addition, a platelet hyperactivity state, characterized by increased agonist (arachidonic acid, ADP and collagen) induced platelet aggregation, platelet expression of adhesion molecules (P-selectin and GP IIb/IIIa), and platelet intracellular calcium concentration, was indicated. Cardiotonic pills showed a significant antioxidative activity by presenting an increase in plasma superoxide dismutase and decrease in erythrocyte glutathione, as well as a lipid-lowering effect (decrease in both plasma cholesterol and triglyceride). Either aspirin or cardiotonic pills could significantly reverse the platelet hypersensitivity and hyperfunction. Compared with aspirin, cardiotonic pills showed a more exaggerated inhibitory effect on platelet function (a significantly decreased collagen-stimulated platelet aggregation, and expression of adhesion molecules). In conclusion, cardiotonic pills inhibited platelet hyperfunction in dogs with insulin resistance. This inhibitory effect may mainly be explained by antioxidative activity and metabolic control.

Short-term, Moderate Dosage Vitamin E Supplementation May Have No Effect on Platelet Aggregation, Coagulation Profile, and Bleeding Time in Healthy Individuals

To investigate the in vivo effect of short-term, moderate dosage synthetic dl-alpha-tocopherol acetate supplementation on platelet aggregation, coagulation profile, and simulated bleeding time in healthy individuals. alpha-tocopherol is the most biologically active isomer of Vitamin E, traditionally promoted as an antioxidant and therapeutic agent in cardiovascular disease. In vitro studies have suggested that alpha-tocopherol plays a role in the inhibition of platelet aggregation. However, further investigations into the effect of alpha-tocopherol on bleeding in vivo have not duplicated these findings. A total of 42 healthy volunteers complied with a 2-week abstinence period from the use of anti-platelet agents followed by determination of baseline platelet aggregation properties and coagulation studies using citrated whole blood. Moderate dosage Vitamin E (800 IU of dl-alpha-tocopherol acetate) was then self-administered for 14 days with reevaluation of platelet aggregation and coagulation profile, and simulated bleeding time after 14 days of Vitamin E supplementation. Forty subjects completed the 4-week study period. All 40 subjects demonstrated normal baseline coagulation studies and all had collagen-stimulated platelet aggregation assessment performed in triplicate. After Vitamin E supplementation, no significant difference was demonstrated in any study parameter. Dietary supplementation with moderate dosage synthetic dl-alpha-tocopherol acetate did not significantly prolong bleeding or platelet aggregation in vivo. The affect of Vitamin E on platelet aggregation in vitro does not appear to be reproducible in vivo. Therefore, peri-operative discontinuation of Vitamin E may not be necessary.

Naproxen Has No Relevant Effect on the Safety, Tolerability, Pharmacodynamics, and Pharmacokinetics of BAY 59-7939 - An Oral, Direct Factor Xa Inhibitor.

BAY 59-7939 is a novel, oral, direct Factor Xa inhibitor in clinical development for the prevention and treatment of thromboembolic disorders. BAY 59-7939 compared favourably with enoxaparin in recent clinical trials of the prevention of venous thromboembolism following major orthopaedic surgery. Possible concomitant medications in patients receiving BAY 59-7939 for either the prevention or treatment of venous thromboembolism include non-steroidal anti-inflammatory drugs (NSAIDs), such as naproxen. This randomized, two-way crossover study was performed to investigate the influence of naproxen on the safety, tolerability, pharmacodynamics, and pharmacokinetics of BAY 59-7939 in 11 healthy male subjects. The study included a run-in period with naproxen. Treatments were: naproxen 500 mg on 2 consecutive days (run-in), followed by a 14-day washout period, and then randomization either to BAY 59-7939 15 mg; or to naproxen 500 mg on the first day, and naproxen 500 mg and BAY 59-7939 15 mg on the second day. There was a 14-day washout period between crossovers. BAY 59-7939, naproxen, and the combination were well tolerated. Adverse events (eight in total) were reported by three subjects, and all were mild in intensity; there were no drug-related, treatment-emergent adverse events. BAY 59-7939 significantly inhibited Factor Xa activity by 35%, and prolonged prothrombin time (by 1.4 times baseline [tb]), activated partial thromboplastin time (1.3 tb), and HepTest (1.9 tb), with no influence from naproxen. No interaction was observed with respect to collagen-stimulated platelet aggregation. BAY 59-7939 and naproxen together significantly increased bleeding time compared with BAY 59-7939 alone; however, this difference was small compared with naproxen alone for all but one subject. This indicates that some subjects may be more sensitive to the combined effect of naproxen and BAY 59-7939. However, analysis of patients' data from clinical trials after major orthopaedic surgery showed similar bleeding risks in patients with and without co-medication with NSAIDs at BAY 59-7939 doses up to 10 mg twice daily. The area under the plasma concentration-time curve (AUC) and maximum plasma concentration (Cmax) for BAY 59-7939 both increased by approximately 10% following co-administration of naproxen; however, this small increase in BAY 59-7939 bioavailability was not considered clinically relevant. In conclusion, there is no relevant interaction between BAY 59-7939 and naproxen, although some individuals may be more sensitive to a combination of these drugs. Initial analysis of data from phase II clinical trials of BAY 59-7939 has shown a similar bleeding risk in patients using NSAIDs concomitantly compared with BAY 59-7939 alone. This will be further substantiated in phase III trials.ParameterNaproxenBAY 59-7939BAY 59-7939 + naproxenBleeding time (tb)a1.46/0.5831.20/0.6132.17/0.576Platelet aggregationb0.0521.020.086AUC (μg.h/L)a-1250/28.561396/26.30Cmax (μ g/L)a-152.9/31.51165.3/27.69aGeometric mean/geometric coefficient of variation; bMedian relative change from baseline; N=11 for all data

Function of the cysteine-rich domain of the haemorrhagic metalloproteinase atrolysin A: targeting adhesion proteins collagen I and von Willebrand factor

The cysteine-rich domain of the haemorrhagic metalloproteinase atrolysin A was shown to inhibit collagen-stimulated platelet aggregation and to interact with MG-63 osteosarcoma cells via integrin alpha2beta1 to inhibit adhesion to collagen I. In addition, we demonstrate by solid-phase binding assays that atrolysin A binds to collagen I and to vWF (von Willebrand factor) via exosites in the cysteine-rich domain. Interestingly, the binding site of the cysteine-rich domain on collagen I is distinct from the cell adhesion site, since the incubation of collagen-I-coated plates with the cysteine-rich domain did not prevent the adhesion of MG-63 cells to collagen. Finally, we show by surface plasmon resonance (BIAcore) analyses that the cysteine-rich domain can block vWF binding to collagen I as well as the binding of collagen I to vWF. Taken together, these results indicate that this domain may function as a cell-surface-receptor-binding site and/or a substrate recognition exosite and may thus play a role in the pathologies associated with atrolysin A.

Quercetin inhibits collagen-stimulated platelet activation through inhibition of multiple components of the glycoprotein VI signaling pathway

The regulation of platelet function by pharmacological agents that modulate platelet signaling has proven a successful approach to the prevention of thrombosis. A variety of molecules present in the diet have been shown to inhibit platelet activation, including the antioxidant quercetin. In this report we investigate the molecular mechanisms through which quercetin inhibits collagen-stimulated platelet aggregation. The effect of quercetin on platelet aggregation, intracellular calcium release, whole cell tyrosine phosphorylation and intracellular signaling events including tyrosine phosphorylation and kinase activity of proteins involved in the collagen-stimulated glycoprotein (GP) signaling pathway were investigated. We report that quercetin inhibits collagen-stimulated whole cell protein tyrosine phosphorylation and intracellular mobilization of calcium, in a concentration-dependent manner. Quercetin was also found to inhibit various events in signaling generated by the collagen receptor GPVI. This includes collagen-stimulated tyrosine phosphorylation of the Fc receptor gamma-chain, Syk, LAT and phospholipase Cgamma2. Inhibition of phosphorylation of the Fc receptor gamma-chain suggests that quercetin inhibits early signaling events following stimulation of platelets with collagen. The activity of the kinases that phosphorylate the Fc receptor gamma-chain, Fyn and Lyn, as well as the tyrosine kinase Syk and phosphoinositide 3-kinase was also inhibited by quercetin in a concentration-dependent manner, both in whole cells and in isolation. The present results provide a molecular basis for the inhibition by quercetin of collagen-stimulated platelet activation, through inhibition of multiple components of the GPVI signaling pathway, and may begin to explain the proposed health benefits of high quercetin intake.

Human lipoprotein (a)-induced reduction of platelet aggregation is not mediated by apolipoprotein A's lysine-binding regions.

Intact lipoprotein (a) (Lp (a)) is known to decrease collagen-stimulated platelet aggregation in vitro, though the nature of the interaction between this lipoprotein and stimulated platelets is unknown. Lysine binding regions of Lp (a) facilitate binding between it and at least one cell type. Epsilon aminocaproic acid (EACA) renders Lp (a)'s lysine binding regions incapable of cellular interaction using those regions. Washed human platelets were presented vehicle alone or with varying concentrations of Lp (a) either not exposed or previously exposed to increasing levels of EACA. In all experiments, EACA failed to affect the impact of Lp(a) on platelet aggregation. It is concluded that the lysine binding regions of Lp (a) do not mediate the intact Lp(a)-induced reduction of collagen-stimulated platelet aggregation in vitro.

Apolipoprotein (a) mediates the lipoprotein (a)-induced biphasic shift in human platelet cyclic AMP

Lipoproteins are known to influence platelet cyclic adenosine monophosphate (c-AMP) levels. Lipoprotein (a) (Lp(a))'s impact on platelet c-AMP levels has never been assessed. Increasing levels of purified human Lp(a) (1–100) mg/dl were incubated with washed human platelets. Lp(a) concentrations of 1–25 mg/dl resulted an initial statistically significant increase of platelet c-AMP above basal levels and decreased collagen-stimulated platelet aggregation levels. Higher concentrations progressively returned the platelet c-AMP concentrations to basal levels accompanied by further decreases in platelet aggregation. Increasing concentrations of purified apolipoprotein (a) (apo(a)) also resulted in a similar biphasic c-AMP response while Lp(a) without apo(a) was without impact. One antibody directed against apo(a) in intact Lp(a) removed the biphasic c-AMP pattern and eliminated Lp(a) platelet aggregation. Antibodies directed against apo B in intact Lp(a) gave results similar to intact Lp(a) in terms of the biphasic response of c-AMP upon platelet exposure to increasing levels of Lp(a). It is concluded that apo(a) mediates the Lp(a)-induced biphasic response in platelet c-AMP as the result of platelet exposure to increasing levels of Lp(a). The biphasic response in c-AMP assists in platelet aggregation decreases up to a concentration of 25 mg/dl Lp(a), such assistance being lost at higher Lp(a) concentrations.

Modulation of in vitro platelet 5-HT release by species of Erythrina and Cymbopogon

Extracts of Australian plants were screened to detect constituents affecting adenosine di-phosphate (ADP) induced platelet aggregation and [ 14C]5-hydroxytryptamine (5-HT) release. Extracts of four tested plants including, Eremophila gilesii, Erythrina vespertilio, Cymbopogon ambiguus, and Santalum acuminatum, were found to cause significant inhibition of platelet 5-HT release. Inhibition levels ranged from 56 – 98 %, and was not due to the non-specific effects of protein binding tannins. These extracts, and those we have previously identified as being active, were examined further to determine if they affect epinephrine (EPN), arachidonic acid (A.A) or collagen stimulated platelet aggregation and 5-HT release. Among those extracts investigated, we found that both the methanolic extract of E. vespertilio and the dichloromethane (DCM) extract of C. ambiguus were most potent and caused significant inhibition of platelet activation induced by EPN, A.A and to a lesser extent by collagen. Inhibition of ADP induced platelet 5-HT release by both of these extracts, was dose-dependent, with IC 50 values for E. vespertilio and C. ambiguus estimated to be 20.4 μl (1.855 mg/ml) and 8.34 μl (0.758 mg/ml), respectively. Overall, C. ambiguus exhibited most activity and also caused dose-dependent inhibition of A.A induced platelet activation. These results indicate that inhibition may occur specifically at a site within the A.A pathway, and suggest the presence of a cyclo-oxygenase inhibitor. Both E. vespertilio and C. ambiguus are reported to be traditional headache treatments, with the present study providing evidence that they affect 5-HT release.

Inhibition of Platelet Aggregation by the Recombinant Cysteine-Rich Domain of the Hemorrhagic Snake Venom Metalloproteinase, Atrolysin A

The P-III class of venom metalloproteinases has, in addition to the proteinase domain, a disintegrin-like domain and a cysteine-rich domain. Recent evidence has shown that the nonproteinase domains of the P-III class of hemorrhagic metalloproteinases function in the inhibition of platelet aggregation by blocking essential procoagulant integrins on platelets. A specific role for the highly conserved cysteine-rich domain has yet to be described. In this study, we expressed the cysteine-rich domain from the hemorrhagic metalloproteinase atrolysin A and demonstrated its ability to inhibit collagen-stimulated platelet aggregation. Additionally, the cysteine-rich domain was shown to interact with MG-63 cells to inhibit adhesion to collagen I. These data suggest a functional role for the cysteine-rich domain of the P-III toxins in the observed coagulopathy by targeting the toxin to platelets and inhibiting collagen-stimulated platelet aggregation. These characteristics may function to synergistically increase the hemorrhagic effect of the toxins.

An inhibitor selective for collagen-stimulated platelet aggregation from the salivary glands of hard tickHaemaphysalis longicornis and its mechanism of action

Soluble materials of salivary glands fromHaemaphysalis longicornis were found to inhibit collagen, ADP, and thrombin-stimulated platelet aggregation. One inhibitory component was purified to salivary gland homogeneity by a combination of gel filtration, ion-exchange, and C(8) reverse phase HPLC. The purified activity, named longicomin, is a protein of molecular weight 16 000 on SDS-PAGE under both reduced and nonreduced conditions. Collagen-mediated aggegation of platelets in plasma and of washed platelets (IC(50) was approximately 60 nmol/L) was inhibited with the same efficacy. No inhibition of aggregation stimulated by other effectors, including ADP, arachidonic acid, thrombin, ristocetin, calcium ionophore A23187, thromboxane A2 mimetic U46619 and 12-O-phorbol-13-myristate acetate, was observed. Longiconin had no effect on platelet adhension to collagen. Not only platelet aggregation but also release reaction, and increase of intracellar ca(2+) level of platelets in response to collagen were completely eliminated by longicomin. Increasing amounts of collagen are able to overcome the inhibition of aggregation by longicomin, indicating that longicomin probably shares with collagen a common receptor. In addition, collagen fibers did not emit fluorescence after incubation with isothocyanate-conjugated longicornin, indicating that longicomin did not bind directly to collagen fibers. The identification and isolation of longicomin demonstrates the existence of a new type of platelet inhibitor that should be useful to better undentand the mechanism of collagen stimulation of platelets.

Atherosclerotic aortic gangliosides enhance integrin-mediated platelet adhesion to collagen.

Gangliosides, sialic acid-containing glycosphingolipids, accumulate in atherosclerotic vessels. Their role in the pathogenesis of atherosclerosis is unknown. Gangliosides isolated from tumor cells promote collagen-stimulated platelet aggregation and ATP secretion and enhance platelet adhesion to immobilized collagen. These activities are all mediated by ganglioside effects on the platelet integrin collagen receptor alpha2beta1. Therefore, we hypothesized that gangliosides isolated from atherosclerotic plaques would enhance platelet adhesion to immobilized collagen, a major component of the subendothelial matrix of blood vessels. Furthermore, we questioned whether this effect of atherosclerotic gangliosides might play a role in the pathogenesis of atherosclerosis. To test this hypothesis, we isolated the gangliosides from postmortem aortas of patients with extensive atherosclerotic disease and examined their effects on platelet adhesion. Samples of aortic tissue taken from areas involved with atherosclerotic plaque demonstrated accumulation of gangliosides (64.9+/-6.5 nmol/g wet weight) compared with gangliosides isolated from control normal aortic tissue taken from children who died of noncardiac causes (NAGs; 21.1+/-6.4 nmol/g wet weight). Interestingly, samples of tissue taken from diseased aortas but from areas not involved with gross plaque formation also demonstrated ganglioside accumulation (47.6+/-12.8 nmol/g wet weight). Next, the activity of each of these gangliosides on platelet adhesion to immobilized type I collagen was studied. Atherosclerotic aortic gangliosides (AAGs) as well as those isolated from grossly unaffected areas of the same aorta (UAGs) both increased platelet adhesion compared with control NAGs (OD570, 0. 37+/-0.11 and 0.29+/-0.14 versus 0.16+/-0.07, respectively; P<0.01 and P<0.05, respectively). These OD570 values corresponded to 9x10(5), 8x10(4), and 6x10(3) platelets per well after preincubation with 5 micromol/L AAG, UAG, and NAG, respectively. Increased adhesion was observed after preincubation with as little as 0.5 micromol/L AAG, and maximal adhesion was seen at 2.5 micromol/L, with a plateau extending to the highest concentration tested, 10 micromol/L. The effect of AAGs on platelet adhesion to collagen was abrogated by incubation of treated platelets with F-17 anti-alpha2 monoclonal antibody (OD570, 0.13+/-0.02). Finally, the effects of the major individual gangliosides isolated from atherosclerotic tissues, GM3 and GD3, were tested. GM3 increased adhesion to collagen (OD570, 0.415+/-0.06) as did GD3 (0.31+/-0.08). Similar to that of AAGs, the effect of both molecules was blocked by F-17 (0. 09+/-0.04 and 0.13+/-0.06, respectively). These experiments demonstrate that accumulated atherosclerotic gangliosides promote platelet adhesion to collagen, the major component of the subendothelial matrix. Furthermore, this activity is mediated by an effect of the gangliosides on the collagen-binding integrin alpha2beta1. This activity may provide a mechanism for the development of platelet thrombi at sites where atherosclerotic gangliosides accumulate and help to explain the role of platelets in the process of atherosclerotic disease progression.

Effects of a Normalized Protein Catabolism Rate on Platelet Functions in Non-Dialyzed Uraemic Patients

NPCR (p < 0.001, p < 0.001), lower platelet adhesion rates (p < 0.001, p < 0.001) and a more prolonged bleeding time (p < 0.05, p < 0.01) than the other groups. The patients in the high-BMI group had a lower NPCR than the normal-BMI group (p < 0.01), and their ADP-, epi-nephrine, ristocetin- and collagen-stimulated mean percent platelet aggregation values were higher than those of the low-BMI group (p < 0.01, p < 0.0001, p < 0.01, p < 0.01, respectively) and than those of the normal-BMI group for only epinephrine, ristocetin and collagen (p < 0.001, p < 0.05, p < 0.05, respectively). In low-BMI patients, ADPand epinephrine-stimulated percent aggregation values were lower than those of the nor-mal

Significance of Platelet Serine/ Threonine Phosphatases 1/2A for Storage of Platelet Concentrates

Inhibition of phosphatases 1/2A (ppl/2A) by okadaic acid (OA) prevents thrombin-or collagen-stimulated platelet aggregation, serotonin and ATP release, as well as intracellular Ca2+ increase. OA is added to 12 platelet concentrates (PCs) prepared from 500 ml whole blood. Microcystin LR (MC), a further ppl/2A inhibitor that does not penetrate the cell membrane, is added to 9 PCs. 9 PCs remain untreated. Storage of PCs is carried out on a horizontal agitator for 7 days. Samples are taken daily to estimate WBC, RBC, PLT, cAMP, cGMP and serotonin. 12 h after OA addition, a dose-dependent drop in platelet counts is seen, accompanied by a corresponding increase of particles with sizes of WBC and RBC. Increased WBC and RBC counts are artefacts brought about by platelet aggregation. From day 4 of storage, serotonin is released massively, cAMP consumption is reduced or stopped, and cGMP concentration increases slightly. In untreated and MC-treated PCs no similar reactions are observed. It seems that the inhibition of extracellular ppl/2A (ectophosphatases) prevents platelet activation. The inhibition of intracellular ppl/2A (endophosphata-ses) presumably enhances platelet activation. The different results between OA- and MC-treated PCs lead to these conclusions because, depending on dose and time, OA penetrates platelet membranes, MC does not. Therefore the results of the study give rise to the suspicion that ppl/2A control two sites of signal transduction in platelets of stored PCs: extracellular ppl/2A are included in platelet activation, while intracellular ppl/2A mediate inhibition of activated platelets. Inhibition of ectophosphatases and activation of endophosphatases probably facilitate PC storage.

The effects of doxazosin on platelet aggregation, platelet adhesion and blood coagulation in cynomolgus monkeys

The effect of doxazosin, a selective alpha-1 adrenergic inhibitor, on hemostasis was investigated in 9 cynomolgus monkeys. During 12 weeks of doxazosin treatment (1 mg/kg per day), serum lipids, lipoprotein cholesterols, blood coagulation, platelet aggregation and template bleeding times were measured and compared with predrug values. In addition, platelet adhesion to cultured human umbilical vein endothelial cells (HUVEC) in the presence or absence of doxazosin was evaluated. Platelet aggregation was also determined in monkeys following chronic oral exposure to aspirin (162 mg/day). Doxazosin administration was associated with significant reductions in serum total cholesterol (TC) (−16%) and low density lipoprotein cholesterol (LDL-C) (−23%), while high density lipoprotein cholesterol (HDL-C) levels increased 66%. Doxazosin did not alter any parameters of blood coagulation measured; however, bleeding times were increased significantly (33%) in doxazosin-treated animals. Although collagen-stimulated platelet aggregation was not influenced by either chronic doxazosin or aspirin treatment, the maximal extent of ADP-stimulated platelet aggregation was significantly reduced (−26% and −18%, respectively) compared with the control monkeys. Platelets from untreated control animals displayed reductions in the extent of ADP-stimulated aggregation of 13% and 23%, respectively, when incubated in vitro with 200 and 300 μg/ml of doxazosin. Additionally, the decrease in aggregation response of platelets obtained from doxazosin-treated monkeys was accompanied by a rapid reversal of platelet aggregation. Adhesion to HUVEC by platelets isolated from doxazosin-treated animals was significantly decreased; however, adhesion was not altered when platelets from untreated control animals were incubated with HUVEC in the presence of doxazosin. Thus, the ex vivo and in vitro studies reported in this communication suggest that doxazosin administration to nonhuman primates is associated with beneficial alterations in plasma lipids, platelet aggregation, bleeding times and platelet adhesion to endothelial cells, parameters which are thought to influence risk of cardiovascular disease in both animals and humans.

The effect of borage oil supplementation on human platelet aggregation, thromboxane B 2, prostaglandin E 1 and E 2 formation

The effect of borage oil supplementation on collagen-induced platelet aggregation and pro-aggregatory thromboxane A 2 (TxA 2), prostaglandin E 2 (PGE 2) and anti-aggregatory prostaglandin E 1 (PGE 1) formation was evaluated. Male volunteers consumed, on average, 5.23 g of gamma-linolenic acid (GLA, 18:3n-6) (as borage oil) daily for 42 days followed by a 42 day wash-out period. The increased platelet aggregation at day 22 and 43 was not accompanied by a statistically significant rise in TxA 2, PGE 1, and PGE 2 formation or in their ratios despite the significantly increased dihomo-gamma-linolenic acid (DGLA, 20:3n-6) arachidonic acid (AA, 20:4n-6) ratio in the platelet phospholipids. These observations suggest that the enhanced platelet aggregation may be mediated by GLA, some other component of borage oil or by a non-eicosanoid mechanism. Another experiment suggested high density lipoprotein (HDL) and low density lipoprotein (LDL) from borage oil consumers did not affect collagen-stimulated autologous platelet aggregation and TxA 2 formation.

Isolation of an inhibitor selective for collagen-stimulated platelet aggregation from the soft tick Ornithodoros moubata.

Soluble extracts from the soft tick Ornithodoros moubata were found to inhibit collagen-, ADP-, and thrombin-stimulated platelet aggregation. One inhibitory component was purified to homogeneity by a combination of gel filtration, ion-exchange, and reverse phase high pressure liquid chromatography. The purified activity, named moubatin, is a protein of molecular weight 17,000 and it inhibits the aggregation of washed human platelets stimulated by collagen with an IC50 of approximately 50 nM in the standard assay. At a concentration of moubatin that maximally inhibited collagen-stimulated platelet aggregation, no inhibition of aggregation initiated by other effectors, including arachidonic acid, thrombin, ristocetin, and the calcium ionophore A23187, was observed. Moubatin also inhibits collagen-dependent aggregation in plasma. At a higher concentration of moubatin (> 1 microM) it was also possible to demonstrate an inhibitory effect on the final extent of aggregation induced by a low concentration of ADP. Although moubatin selectively inhibits platelet activation by collagen, it has only a minimal effect on the adhesion of platelets to collagen. The amino acid sequences of peptides derived from proteolytic cleavage of moubatin suggest that moubatin is a unique protein, consistent with its novel functional activity.

An inhibitor of collagen-stimulated platelet activation from the salivary glands of the Haementeria officinalis leech. I. Identification, isolation, and characterization.

A protein that blocks collagen-stimulated platelet aggregation has been identified and isolated from the soluble fraction of salivary glands from Haementeria officinalis leeches. We have named this protein leech antiplatelet protein (LAPP). LAPP was isolated from soluble crude salivary gland extract by heparin-agarose, size exclusion, and C18 reverse phase high-performance chromatography. Its molecular weight is approximately 16,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reduced and nonreduced conditions. The sequences of peptides generated by V8 digestion of LAPP as well as its amino acid composition suggested no homology to other known proteins. The IC50 for LAPP to inhibit platelet aggregation was approximately 60 nM. This inhibitory activity is specific for collagen-induced aggregation. Platelet aggregation in response to ADP, arachidonic acid, U46619, thrombin, and ionophore A23187 was not inhibited by LAPP at a concentration that blocked platelet aggregation to collagen by 100%. In contrast, crude salivary gland-soluble extract contained activity(ies) which inhibited aggregation to all these agonists except thrombin at 1 unit/ml and 2 microM A23187. Thus, the H. officinalis leech has evolved multiple mechanisms to prevent hemostasis, including an inhibitor of collagen-stimulated platelet aggregation. The identification and isolation of LAPP demonstrates the existence of a new type of platelet inhibitor that should be useful to better understand the mechanism of collagen stimulation of platelets.

An inhibitor of collagen-stimulated platelet activation from the salivary glands of the Haementeria officinalis leech. II. Cloning of the cDNA and expression.

Salivary glands of the leech Haementeria officinalis contain a protein, leech antiplatelet protein (LAPP), that specifically blocks collagen-mediated platelet aggregation (Connolly, T. M., Jacobs, J. W., and Condra, C. (1992) J. Biol. Chem. 267, 6893-6898). Degenerate oligonucleotides whose sequences were derived from two short peptides from V8 digests of the native LAPP were used as primers to generate a polymerase chain reaction (PCR) product which contains the cDNA region coding for the sequence between these two peptides. Using this PCR product as a hybridization probe, phage containing cDNA clones were isolated containing the entire deduced amino acid sequence for LAPP. Computer analysis of the amino acid sequence predicts a peptidase cleavage site between a 21-residue pre-peptide and a mature protein of 126 amino acids. A DNA insert to express the predicted mature LAPP protein was generated by PCR amplification using phage-derived cDNA clones as a substrate. This insert encoded a fusion protein with the leader sequence of the yeast alpha mating factor and the mature LAPP cDNA. These PCR products were cloned into the yeast expression vector pKH4 alpha 2. A KEX 2 Lys-Arg endopeptidase cleavage site was placed NH2-terminal to the predicted mature protein. This vector transfected into the yeast Saccharomyces cerevisiae directs expression of a secreted mature protein at levels up to 200 mg of LAPP/liter of culture medium. The recombinant protein was comparable to native LAPP in its electrophoretic mobility, its reactivity with anti-LAPP antisera, and its biological activity including inhibition of collagen-stimulated platelet aggregation and the adhesion of platelets to collagen. Availability of significant quantities of recombinant LAPP opens the way to further biochemical structure/function studies and to studies on the effects of an inhibitor of collagen-stimulated platelet aggregation in vivo.

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.

The effect of endothelium-derived nitric oxide on ex vivo whole blood platelet aggregation in man.

The effects of changes in endogenous endothelium-derived nitric oxide (NO) on forearm blood flow and ex vivo platelet aggregation have been studied in 7 healthy volunteers. Measurements were made of forearm blood flow and ex vivo collagen-stimulated platelet aggregation during unilateral brachial artery infusions of saline, acetylcholine (ACh), NG monomethyl-L-arginine (L-NMMA), and prostacyclin (PGI2). The uninfused arm acted as a control. Forearm blood flow was increased by ACh, an agent which stimulates NO release, and decreased by L-NMMA, an agent which stereospecifically inhibits NO synthesis. Collagen-stimulated platelet aggregation measured ex vivo in whole blood draining the infused arm was unaltered by either ACh or L-NMMA. Conversely, PGI2, an agent which acts independently of NO, caused an increase in forearm blood flow which was accompanied by significant inhibition of platelet aggregation. The results suggest that release of endothelium-derived NO in quantities sufficient to cause substantial changes in blood vessel tone does not lead to changes in platelet aggregation in the blood flowing through the vessels. It is, however, still possible that endothelium-derived NO modulates platelet activity at the level of the endothelium.