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PubMed1995

Regulation of platelet aggregation by post-fibrinogen binding events. Insights provided by dithiothreitol-treated platelets.

A variety of data suggest that fibrinogen binding is necessary but not sufficient for platelet aggregation: post fibrinogen binding events may play an important role. The present study compared fibrinogen binding and platelet aggregation in response to dithiothreitol (DTT) and ADP. DTT induced saturable and specific fibrinogen binding (Kd 0.07 + 0.02 microM, Bmax 15,000 + 3000 molecules/platelet) which supported complete platelet aggregation as determined by single platelet counting. The aggregates were small, however, and more readily dissociated by EDTA than their ADP-treated counterparts, despite quantitatively similar fibrinogen binding. Unlike fibrinogen bound to ADP-stimulated platelets, fibrinogen bound to DTT-treated platelets remained sensitive to dissociation by EDTA over a 3 h time course, retained its ability to support aggregation, even when aggregation was induced 60 min after the initial platelet exposure to fibrinogen, and remained accessible to polyclonal antibodies and plasmin. Confocal scanning laser microscopy showed only a surface clustering of fibrinogen bound to DTT-treated platelets over the 3 h time course compared to rapid fibrinogen clearing from the surface of ADP-stimulated platelets. These data suggest that post fibrinogen binding events involved in the stabilization of fibrinogen binding and/or the redistribution of bound fibrinogen may play important roles in regulating platelet aggregation.

Adenosine Diphosphate↗
PubMed2001

Blockade of adenosine diphosphate receptors P2Y(12) and P2Y(1) is required to inhibit platelet aggregation in whole blood under flow.

Using heparinized whole blood and flow conditions, it was shown that adenosine 5'-diphosphate (ADP) receptors P2Y(12) and P2Y(1) are both important in direct shear-induced platelet aggregation and platelet aggregation subsequent to initial adhesion onto von Willebrand factor (vWf)-collagen. In the viscometer, whole blood was subjected to shear rates of 750, 1500, and 3000 s(-1) for 30 seconds at room temperature. The extent of aggregation was determined by flow cytometry. The P2Y(12) antagonist AR-C69 931MX (ARMX) reduced shear-induced aggregation at these rates by 56%, 54%, and 16%, respectively, compared to control samples. Adenosine 3',5'-diphosphate (A3P5P; P2Y(1) antagonist) inhibited shear-induced aggregation by 40%, 30% and 29%, respectively, compared to control samples. Blockade of both ADP receptors at 3000 s(-1) with ARMX plus A3P5P further reduced the platelet aggregation by 41% compared to the addition of ARMX alone (57% compared to control samples). Using a parallel-plate flow chamber, whole blood was perfused over bovine collagen type 1 at a wall shear rate of 3000 s(-1) for 60 seconds. Platelet deposition was quantified with epifluorescence video microscopy and digital image processing. Blockade of P2Y(12) alone or blockade of P2Y(1) alone did not reduce thrombus formation on vWf-collagen. In contrast, blockade of both P2Y(12) and P2Y(1) reduced platelet deposition by 72%. These results indicate that combinations of antagonists of the ADP receptors P2Y(12) and P2Y(1) are effective inhibitors of direct shear-induced platelet aggregation and of platelet aggregation subsequent to initial adhesion under flow conditions. Inhibitors of these pathways are potentially useful as antiarterial thrombotic agents.

Adenosine Diphosphate↗
PubMed1973

Collagen-mediated platelet aggregation. Effects of collagen modification involving the protein and carbohydrate moieties.

In an effort to elucidate the nature of the collagen-platelet interaction, the effects of collagen modification on platelet aggregation have been studied. We have shown that purified rat skin (salt) soluble collagen is effective at about 20 nM in mediating platelet aggregation in human platelet-rich plasma. This concentration is somewhat greater than that required of several skin insoluble collagens (ca. 10 nM). Both the alpha1(I) and alpha2 chains from rat skin soluble collagen produced platelet aggregation, but only at concentrations of about 13 muM and 55 muM, respectively. In contrast, heat-denatured collagen and chains (e.g., 65 muM alpha1(I) and 160 muM alpha2) failed to induce platelet aggregation and to inhibit platelet aggregation by native collagen. Glycopeptides were prepared from human skin insoluble collagen by extended digestion with bacterial collagenase and trypsin, and were purified by gel filtration into two classes. One class of higher molecular weight contained sialic acid, glucosamine, galactosamine, fucose, mannose, galactose, and glucose, and the other of lower molecular weight consisted primarily of a mixture of galactose and galactosyl-glucose units O-glycosidically linked to hydroxylysine-containing peptides. We found that, after the residual tryptic activity contaminating the higher molecular weight fraction was inhibited, neither of the glycopeptide classes produced nor inhibited native human skin insoluble collagen-mediated platelet aggregation at the highest concentration examined (ca. 1-2 mg glycopeptide per ml of platelet-rich plasma). Highly purified samples of the hydroxylysyl glycosides, hydroxylysylgalactose and hydroxylysylgalactosylglucose (Hyl-Gal and Hyl-Gal-Glc, respectively), were prepared from human urine and labeled at galactose using galactose oxidase followed by reduction with tritiated borohydride. Binding studies with platelet-rich plasma showed that, at concentrations greater than 50 nM, Hyl-Gal gives apparent binding to platelets, but there was no evidence of Hyl-Gal-Glc binding to platelets at concentrations up to 250 nM. At concentrations several hundredfold higher than the equivalents present in the minimum concentration of rat skin soluble collagen required for platelet aggregation, neither Hyl-Gal (at 29 muM) nor Hyl-Gal-Glc (at 18 muM) caused platelet aggregation or inhibited platelet aggregation by native collagen. Also, at a concentration of 85 muM (which represents a concentration about two thousandfold higher than the equivalents in the minimum concentration of soluble collagen required for platelet aggregation) the Gal-Glc-containing 36 residue rat skin soluble collagen alpha1(I)cyanogen bromide #5 peptide had no platelet aggregating or inhibiting activity. Modification of at least 90% of the rat skin soluble collagen carbohydrate by mild periodate oxidation had no effect on the platelet aggregating activity. Human skin insoluble collagen was reacted with periodate under the same conditions, and this had no demonstrable effect on its ability to induce platelet aggregation. This indicates that the normal carbohydrate side chains of these collagens are not required for the platelet interaction that produces the release of ADP and other metabolic constituents and leads to aggregation.Thus, collagen-platelet interactions appear to involve at least two distinct binding sites on the platelet plasma membrane. One is a protein binding site that activates platelet aggregation and has high specificity and affinity for the collagen triple-helical fold or perhaps even for a particular amino acid sequence in the triple helix.

Amino Acids↗
PubMed1990

A method for the determination of circulating aggregated platelets and its application to patients in the course of unstable angina.

A method for the quantitative and qualitative determination of the number of aggregated platelets is described. One milliliter of venous blood was separated equally into two solutions. One solution composed of EDTA (ethylenediaminetetraacetic acid) and formaldehyde (solution F) contained reversibly and irreversibly aggregated platelets, and the second solution, composed of EDTA alone (solution E), contained irreversibly aggregated platelets. By microscopic readings, the percentage of platelets forming aggregates was determined. Reversibly aggregated platelets were estimated by subtracting the percentage of aggregated platelets in solution E from that in solution F. The average amount of platelets per aggregate was calculated by dividing the number of aggregated platelets in solution F by the number of aggregates per 1000 platelets counted. The reference ranges (means +/- SDs) established in 100 healthy persons were 5.8% +/- 2.4% (1% to 9%) for solution F, 3.9% +/- 1.8% (0% to 7%) for solution E, and 2.2 +/- 0.18 (2.0 to 2.5) for the average number of platelets per aggregate. Twenty hospitalized patients without heart disease had values similar to those of 100 normal subjects. In 50 patients with acute myocardial infarction, the percentage of aggregated platelets in solution F was 23.8% +/- 10.3%; in solution E, 4.0% +/- 3.0%; and the average number of platelets per aggregate, 2.9 +/- 0.7. The mean variance for five daily consecutive measurements was 0.52% for solution F, 0.63% for solution E, and 0.002 for the average number of platelets per aggregate. An even lesser mean variance was observed when the interobserver-vs-intraobserver and the intersmear-vs-intrasmear variations were tested. In patients with acute myocardial infarction, the interobserver-vs-intraobserver variance was 5.6% for solution F, 2.2% for solution E, and 0.005 for the average number of platelets per aggregate. The parameters studied were unaffected by different blood drawings, assay tubes, or venous stasis. In 80 patients with unstable angina, the studied parameters as well as the percentage of "big" platelets were measured on hospital days 1, 2, and 5. In 25 patients in whom acute myocardial infarction developed during hospitalization, the percentage of aggregated platelets was 28.1% +/- 8.3%. Most of them (71%) were reversibly aggregated and did not change during hospitalization. The average number of platelets per aggregate was 3.9 +/- 1.6, and the percentage of big platelets was 12.5% +/- 7.2%, both values not undergoing subsequent changes. In patients in whom acute myocardial infarction did not develop, the percentage of aggregated platelets decreased to 14.2% +/- 6.1% on day 5. Most aggregated platelets (58.8% to 90%) were irreversibly aggregated.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1993

Effect of garlic on platelet aggregation in patients with increased risk of juvenile ischaemic attack.

A platelet-inhibiting effect is described for garlic. In this double-blind, placebo-controlled study on 60 voluntary subjects with cerebrovascular risk factors and constantly increased platelet aggregation it was demonstrated that the daily ingestion of 800 mg of powdered garlic (in the form of coated tablets) over 4 weeks led to a significant inhibition of the pathologically increased ratio of circulating platelet aggregates and of spontaneous platelet aggregation. The ratio of circulating platelet aggregates decreased by 10.3%, from 1.17 +/- 0.08 to 1.05 +/- 0.11 (P < 0.01), and spontaneous platelet aggregation by 56.3%, from 40.7 +/- 23.3 to 17.8 +/- 23.2 degrees (P < 0.01) during the garlic phase. There were no significant changes in the placebo group. The parallel group comparison (garlic versus placebo) revealed a significantly different ratio of circulating platelet aggregates after 4 weeks of treatment (P < 0.05). After the 4-week wash-out phase the values increased again to 1.19 +/- 0.32 and 34.9 +/- 28.7 degrees, reaching the initial values (run-in phase prior to the ingestion of garlic). Since garlic is well tolerated it would be worth testing it in a controlled clinical trial for usefulness in preventing disease manifestations associated with platelet aggregation.

Brain Ischemia↗
PubMed1989

Blockade of ADP-induced Ca2+-signal and platelet aggregation by lipoxygenase inhibitors.

Stimulation of platelets results in the liberation of arachidonic acid (AA) which is further metabolized via the cyclooxygenase or lipoxygenase (LPG) pathway. We have examined the effect of inhibition of LPG on (i) the ADP-induced increase of cytoplasmic Ca2+ concentration and (ii) platelet aggregation. Lipoxygenase inhibitors, nordigidroguaiaretic acid (NDGA) and BW-755C, both suppressed ADP-induced Ca2+-signals and aggregation in a dose-dependent manner, with an IC50 value of 1 2 microM for NDGA. Qualitatively the same effect was obtained with 4-bromophenylacyl bromide, the inhibitor of phospholipases A2 and C. By contrast, cyclooxygenase inhibitor indomethacin had only a negligible effect on Ca2+-signals and suppressed only the second phase of ADP-induced aggregation. It is concluded that the LPG pathway of AA metabolism in platelets might play a crucial role in ADP-induced Ca2+-signal generation and platelet aggregation.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
PubMed1989

Heparin-associated thrombocytopenia: observations on the mechanism of platelet aggregation.

We investigated the mechanism of heparin-mediated platelet aggregation in 11 patients with heparin-associated thrombocytopenia. Severe thrombocytopenia (16,000 to 66,000 platelets/microliters) developed in each patient during heparin therapy, and platelet aggregation occurred in vitro when heparin was added to mixtures of patient plasma and normal platelet-rich plasma. In 10 patients, heparin-initiated platelet aggregation was inhibited by preincubation of mixtures of normal platelet-rich plasma and heparin-associated thrombocytopenia plasma with monoclonal antiglycoprotein Ib antibodies 6D1 or LJ-Ib1. Both antibodies are directed against the von Willebrand factor binding site on glycoprotein Ib and inhibit only ristocetin-induced platelet agglutination. Purified immunoglobulin G (IgG) from patients with heparin-associated thrombocytopenia also supported heparin-induced aggregation, but equivalent amounts of antigen-binding fragments [F(ab')2] did not. We also found that F(ab')2 of LJ-Lb1 did not inhibit heparin-induced platelet aggregation but retained inhibitory activity against ristocetin-induced platelet agglutination. The monoclonal antibody 3G6, directed against the alpha-chain of glycoprotein Ib but not inhibitory of ristocetin-induced platelet agglutination, had no effect on heparin-induced platelet aggregation. Antibodies to von Willebrand factor that inhibit ristocetin-induced platelet agglutination did not inhibit heparin-mediated platelet aggregation, but antibodies to glycoprotein IIb-IIIa blocked aggregation. These data suggest that platelet aggregation in heparin-associated thrombocytopenia may be initiated by an interaction between patient IgG, heparin, and the platelet surface. Platelet activation appears to be mediated by a platelet surface crystallizable fragment (Fc) receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Heparin↗
PubMed1996

Inhibition of PAF-induced platelet aggregation by WEB 2086 'in-vitro', an antagonist to the receptor for platelet-activating factor, in bovine.

The sensitivity of bovine platelet aggregation in response to PAF stimulation and the ability of WEB 2086 (a thieno-triazolodiazepine) to inhibit response to PAF-induced platelet aggregation were investigated in the blood from five healthy male Belgian Blue calves. The recorded response to PAF showed a plateau which was dependent on the PAF concentration. Platelet aggregation induced by PAF consists of two mechanisms: reversible and irreversible aggregations which are accompanied by the release of platelet granule contents. Reversible aggregation occurred above (2 . 10(-9) mol/l) PAF, and irreversible aggregation occurred above (2 . 10(-7) mol/l) PAF. Addition of WEB 2086 to bovine platelets in vitro induced a rightward shift in the dose-response curve to PAF. WEB 2086 inhibited PAF-induced aggregation in a competitive reversible manner (pA2 = 7.61). The results of our study show that PAF induces platelet aggregation in platelet-rich plasma (PRP) and that addition of WEB 2086 to bovine platelets in vitro inhibits PAF-induced Platelet Aggregation.

Animals↗
PubMed1983

Effects of acetylsalicylic acid on platelet aggregation before and during increase in dietary eicosapentaenoic acid.

The effect of acetylsalicylic acid (ASA) on platelet aggregation before and during a fish diet, already known to decrease the aggregability of platelets and to prolong the bleeding time, was studied in 10 healthy men. Two doses (3.5 and 10 mg/kg body weight) of ASA were given. Both doses equally decreased platelet aggregation to collagen and adenosine diphosphate (ADP). ASA, taken before the diet, diminished platelet aggregability to ADP by as much as did the diet alone. When ASA was administered during the diet, the effect on platelet aggregability to ADP was additive. Aggregation to collagen also decreased to the same extent as during the baseline period. The results, in conjunction with our earlier ones, indicate that the mechanism by which a fish diet delays primary haemostasis is different from the apparently similar effect of ASA. This raises the possibility of augmenting any antithrombotic effect of ASA by dietary means.

Adenosine Diphosphate↗
PubMedDate not supplied

The effect of glycosylated albumin on platelet aggregation.

BACKGROUND: Albumin has a role in the complicated process of platelet aggregation. Although it is known that quantitative changes in plasma albumin alter platelet aggregation, less is known about the interaction between qualitative changes in plasma albumin and platelets. One common qualitative change in plasma albumin is nonenzymatic glycosylation, which occurs during states of prolonged hyperglycemia. METHODS: Albumin was selectively removed from normal plasma by means of an affinity column. Glycosylated albumin was added to this albumin-poor plasma, and it was used to study platelet aggregation induced by low concentrations of arachidonic acid. Platelet aggregation was determined by light transmittance. RESULTS: As the concentration of glucose in which albumin was incubated was increased, there was progressive augmentation of platelet aggregation. At a plasma glucose of 150 mg/dL, average light transmittance was 9.4%, and at 200 mg/dL, it was 24.6%. These values were significantly different at a p value < .01. At glucose levels of 300 mg/dL and 400 mg/dL, mean light transmittance was 40.6% and 74.4%, respectively, and these values were significantly different with p values of < .01. CONCLUSIONS: Platelet aggregation in response to a relatively low concentration of arachidonic acid is enhanced in the presence of albumin that has been incubated in a medium containing levels of glucose that are higher than would be seen in normal patients but are consistent with those seen in diabetics with less than optimal control. This augmentation of platelet aggregation is statistically significant.

Adenosine Diphosphate↗
PubMed1986

von Willebrand factor contaminating porcine factor VIII concentrate (Hyate:C) causes platelet aggregation.

Severe thrombocytopenia has been reported to occur in some patients treated with a polyelectrolyte-fractionated porcine factor VIII concentrate (Hyate:C) (Kernoff et al, 1984; Gatti & Mannucci, 1984). We report here that Hyate:C induces aggregation of platelet-rich plasma in vitro accompanied by ATP release and thromboxane B2 formation, and is inhibited by EDTA and prostaglandin E1. Hyate:C induced platelet aggregation is affected by monoclonal antibodies anti-glycoprotein Ib or anti-glycoprotein IIb/IIIa and suppressed by polyclonal anti-porcine von Willebrand factor antiserum. We conclude that the aggregating ability of Hyate:C is due to porcine von Willebrand factor present in the concentrate. The occasional thrombocytopenia described after Hyate:C infusions probably results from the interaction between platelets and the porcine von Willebrand factor contaminating the concentrate.

Adenosine Triphosphate↗
PubMed1999

Platelet aggregation may not be a prerequisite for collagen-stimulated platelet generation of nitric oxide.

By determining the sum of the supernatant concentrations of nitrite and nitrate the stimulated generation of nitric oxide (NO) by human washed platelets induced by a range of fibrillar collagen concentrations (0.0156-25 microg ml(-1)) was investigated. Platelet serotonin (5-hydroxytryptamine, 5-HT) efflux and platelet aggregation were also measured. Under resting conditions (0 microg ml(-1) collagen) platelet NO release was equivalent to 1.06+/-0.17 nmol per 10(8) platelets. Maximal NO release, equivalent to 2.1+/-0. 37 nmol per 10(8) platelets, was observed with only 0.0625 microg ml(-1) collagen (P<0.02, stimulated vs. resting release), higher collagen concentrations producing no further increases in platelet NO output. By contrast, maximal platelet aggregation and 5-HT efflux did not occur until collagen concentrations of 2.5 microg ml(-1) and 10-25 microg ml-1), respectively, had been achieved. L-NAME (1 mmol l(-1)) and L-NMMA (1 mmol l(-1)) inhibited stimulated platelet NO generation by 78+/-6% and 72%, respectively. Contrasting with fibrillar collagen, fibrillar beta-amyloid protein had no effect on platelet NO generation, or on 5-HT efflux or aggregation. These data perhaps indicate that NO generation by human platelets is stimulated by concentrations of fibrillar collagen insufficient to elicit an aggregatory response. Such a mechanism could operate in vivo to inhibit platelet aggregation which might otherwise be induced by low concentrations of circulating agonists.

Blood Platelets↗
PubMedDate not supplied

Potentiation of endoperoxide analog-induced platelet aggregation by heparin.

The mechanism of heparin-induced platelet aggregation potentiation is much debated. We examined the effects of heparin on platelet aggregation induced by cyclic endoperoxide analog u46, 619. Heparin enhanced platelet aggregation in a dose-related fashion. Potentiation of platelet aggregation was also found to be related to the incubation time of heparin and platelet rich plasma. Porcine intestinal heparin and bovine lung heparin exhibited similar patterns. To test the hypothesis of increased platelet thromboxane A2 production under the influence of heparin, we measured U46,619-induced thromboxane A2 generation. Heparin was found not to affect platelet thromboxane A2 generation over a wide range of concentrations. The platelet aggregation potentiating actions of heparin are thus unrelated to thromboxane A2 pathway.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1982

Inhibition of spontaneous platelet aggregation and adhesion by indobufen (K 3920). A randomized, double-blind crossover study on platelet, coagulation and fibrinolysis function tests.

In a randomized double-blind crossover study in 12 patients with atherosclerotic disease, the effect of 2 dosages (100 and 200 mg twice daily) of indobufen, a new synthetic inhibition of platelet aggregation, on some platelet functions, coagulation and fibrinolysis tests was investigated. Regardless of the dosage used, indobufen was shown to induce a prompt normalization of the enhanced platelet aggregation of these patients. The effect lasted for the entire period of drug administration and in 50% of patients a normal platelet aggregation was maintained until the fourth day after discontinuation of the drug. Indobufen was also able to reduce platelet adhesiveness and to lengthen bleeding time, especially when the higher dosage was used.

Aged↗
PubMed1996

Terazosin: ex vivo and in vitro platelet aggregation effects in patients with arterial hypertension.

Antihypertensive effect, platelet aggregation, and plasma lipid profile were studied in a group of 14 hypertensive patients with diastolic blood pressure between 96 and 116 mm Hg during placebo and terazosin phases. Terazosin, an alpha 1-adrenergic blocking agent, was given initially at the dosage of 1 mg daily. Then it was continued at a dosage of 2 mg daily and 5 mg daily respectively, each dosage for 4 weeks. Blood pressure was taken every 2 weeks. Ex vivo platelet aggregation induced by epinephrine, collagen, and adenosine diphosphate (ADP) were carried out twice during the first placebo phase, once at the end of each terazosin dosage, and once in the second placebo phase. Total cholesterol, HDL cholesterol, and triglycerides were measured at the end of first placebo and terazosin phases. Blood from eight patients was taken during the second placebo phase to carry out in vitro response of platelet aggregation induced by ADP, collagen, and epinephrine before and after incubation with terazosin (1, 2 and 5 micrograms/L or doxazosin (100, 200, and 500 micrograms/L for 5 min. Terazosin induced a statistically significant decrease in 14.2/8.0 mm Hg, 26.1/13.4 mm Hg, and 33.9/16.5 mm Hg in the supine position for 1, 2, and 5 mg/daily, respectively. No changes in heart rate were observed. Terazosin inhibited significant ex vivo platelet aggregation induced by epinephrine, collagen, and ADP in a range from 20% to 45% for different concentrations of inducers. Reductions in platelet aggregation seemed not to be dose dependent, as reductions were statistically equivalent for dosages of 1, 2, and 5 mg daily. Terazosin significantly reduced the level of total cholesterol (8.71%) and triglycerides (14.31%), and increased (although not significantly) levels of HDL cholesterol (3.91%). In vitro platelet aggregation was inhibited by doxazosin to a significant extent but not by terazosin.

Adrenergic alpha-1 Receptor Antagonists↗
PubMed1994

Effect of vintoperol on platelet aggregation and experimental thrombosis.

The platelet aggregation inhibitory and antithrombotic effect of the new peripheral circulation enhancing compound vintoperol (RGH-2981, CAS 106498-99-1) was studied. In vitro, vintoperol inhibited the aggregation response to collagen in platelet-rich plasma from mice, rats, rabbits and dogs. It was found to be highly effective in preventing mice from acute pulmonary thromboembolic death induced by adenosine diphosphate (ADP) or collagen. After the oral dose of 10 mg/kg the percentage of survivors increased from 9 to 60% and from 13 to 73%, respectively. In the "mouse antithrombotic assay" it was protective only at the 3 mg/kg dose. Sudden death of mice evoked by hardened red blood cell suspension was not protected by vintoperol. In mice receiving 30 mg/kg vintoperol orally, the inhibition of aggregation response to collagen, ADP and ADP/epinephrine by 15, 33 and 37%, respectively, was associated with a substantial increase in bleeding time. In a rat multifactorial thrombosis model the 10 mg/kg p.o. dose was also sufficient to obtain significant antithrombotic effect (p < 0.01). Results of these experiments indicate that vintoperol interferes with platelet aggregation both in vitro and in vivo and possesses potent antithrombotic effects in thrombosis models in which platelet activation is mainly involved.

Adenosine Diphosphate↗
PubMed1996

Evidence for the involvement of complement proteins in platelet aggregation by Streptococcus sanguis NCTC 7863.

We investigated the mechanisms of platelet aggregation by the type strain of Streptococcus sanguis (NCTC 7863). This species is one of the major aetiological agents of infective endocarditis. S. sanguis NCTC 7863 caused aggregation of normal human platelets in vitro following a lag period that varied between donors (7-19 min). Platelet aggregation was dependent on one or more plasma constituents and all the necessary factors gradually became bound to the bacterial surface during the lag period. The length of the lag period was determined by the plasma of the donor and not by a feature of their platelets. Platelet aggregation by S. sanguis NCTC 7863 could be inhibited by heating plasma at 56 degrees C, by treating plasma with cobra venom factor, or by incubating with soluble Complement Receptor 1, all of which inhibit or deplete complement. Complement activation required Mg2+, but not Ca2+ ions and the the cleavage fragment, Ba, of factor B was produced, indicating that the alternative pathway was operative. Zymosan- and S. sanguis-induced aggregation showed similarities, including the same variability in lag times among donors, and absorption of plasma with zymosan prevented the plasma from supporting platelet aggregation by S. sanguis, C3, C9 and vitronectin were found to bind to S. sanguis NCTC 7863, but the latter two were present at very low levels on a non-aggregating strain of S. sanguis, SK96. The rate of assembly of the C5b-9 complex on the NCTC 7863 bacterial surface correlated with the lag time. These data suggest a role for the complement pathway in platelet aggregation by the type strain of S. sanguis.

Complement C1↗
PubMed2003

Redox control of platelet aggregation.

Sulfhydryl and disulfide metabolism in platelet function has recently reemerged as a focus of platelet research. In this study we tested the effect of redox buffer on platelet aggregation and the effect of reduced glutathione (GSH) and platelet activation on sulfhydryl exposure in the platelet fibrinogen receptor, alpha IIb beta 3. In the presence of subthreshold concentrations of agonist, physiologic concentrations of GSH (10 microM) stimulated platelet aggregation and secretion. These effects were found with more than one platelet agonist and with different low molecular weight thiols, including homocysteine. The effect of low molecular weight thiols was reproduced with the peptide LSARLAF which directly activates platelets through alpha IIb beta 3, suggesting that the mechanism is at the level of this integrin. After determining optimal sulfhydryl labeling conditions for alpha IIb beta 3 (5 mM EDTA, 37 degrees C, 60 min), we found that GSH (10 microM) generated sulfhydryls in the beta 3 subunit. To determine if the requirement was for reducing equivalents or for a redox potential (ratio of GSH to GSSG), aggregation was further studied with the addition of low concentrations of GSSG to the GSH. With a ratio of GSH/GSSG of 5/1, similar to that of blood, the addition of GSSG potentiated the stimulatory effect as compared to GSH alone. This indicates that, for potentiation of aggregation, GSH is not simply reducing disulfide bonds; there is rather a requirement for a certain redox potential. Additional studies performed in the absence of added glutathione showed an increase in sulfhydryl labeling in the beta 3 subunit during platelet activation. Finally, we show that vicinal dithiols of platelet surface proteins are involved in the sulfhydryl-dependent pathways of platelet activation. In summary, these data imply that the redox potential of blood regulates activation of the alpha IIb beta 3 integrin and together with other reports in the literature suggest that disulfide bond cleavage with sulfhydryl generation in beta 3 is involved in activation of this receptor.

Arsenicals↗