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Biomedical subjects

M A Packham

Publications and source records attributed to M A Packham.

At least 37 records · Page 2Linked to original sources
PubMed1996

Probenecid inhibits platelet responses to aggregating agents in vitro and has a synergistic inhibitory effect with penicillin G.

Probenecid is an anion channel blocker and uricosuric agent, originally developed to slow the rate of excretion of penicillin. It is now also administered with many other drugs to reduce their required dosages. Recently, probenecid (2.5 mM) has been used to prevent leakage of fura-2 or fluo-3 when these indicators of cytosolic Ca2+ levels have been introduced into cells. However, we found that probenecid markedly inhibited the increases in cytosolic Ca2+ caused by ADP, thrombin, the thrombin receptor-activating peptide (SFLLRN, TRAP), ADP, sodium arachidonate, the thromboxane A2 (TXA2) mimetic U46619, and platelet-activating factor (PAF). This finding precluded the use of probenecid with platelets in measurements of cytosolic Ca2+ with indicators such as fura-2. We then investigated the effects of probenecid on aggregation and release of 14C-serotonin from prelabeled platelets. Responses to all the agonists were inhibited by 2.5 mM probenecid, but concentrations as low as 0.25-0.5 mM inhibited responses to agonists that act largely via TXA2 (collagen, sodium arachidonate and U46619). Collagen-induced TXA2 formation was inhibited in a dose-dependent manner. Responses of aspirin-pretreated platelets to thrombin, SFLLRN, U46619 and PAF were also inhibited by probenecid, indicating that prevention of TXA2 formation does not account for all the inhibitory effects. The combination of probenecid with penicillin G produced additive or synergistic inhibition of platelet responses; responses dependent on TXA2 were synergistically inhibited by concentrations of the drugs that are reached in vivo. The synergistic inhibitory effect of probenecid on platelet functions could further impair hemostasis if it has already been partially compromised by the administration of other drugs.

Calcium↗
PubMed1995

Effects of thrombin and the thrombin receptor activating peptide, SFLLRN, on redistribution of platelet alpha-granule contents are similar and independent of the extent of thromboxane formation.

Immunocytochemistry with gold-labeled antibodies was used to compare the effects of stimulation of human platelets with thrombin (1 U/ml) and the thrombin receptor activating peptide, SFLLRN (20 microM). After 3 min, redistribution of fibrinogen, von Willebrand factor, and P-selectin (GMP-140, CD62) was examined, the percentages of [14C]serotonin and beta-thromboglobulin released from pre-labeled platelets were measured, and the amount of thromboxane B2 formed was assayed. Upon stimulation with either thrombin or SFLLRN, the platelets had changed from their normal disc shape to spheroidal forms with short pseudopodia. Few alpha-granules remained, the open canalicular system was expanded (more so with SFLLRN) and contained most of the fibrinogen and von Willebrand factor, although small amounts were evident on the platelet surface. Most of the P-selectin was on the surface. Both thrombin and SFLLRN caused complete release of beta-thromboglobulin and 88.3 and 77.5% release of [14C]serotonin, respectively. However, formation of TXB2 caused by thrombin was 10 times greater than that caused by SFLLRN (969 +/- 173 vs 76 +/- 22 ng/10(9) platelets). Thus, the redistribution of platelet alpha-granule contents is similar with thrombin or SFLLRN stimulation and is unaffected by the extent of thromboxane formation.

Amino Acid Sequence↗
PubMed1995

Platelet accumulation on fibrin-coated polyethylene: role of platelet activation and factor XIII.

Platelet accumulation on small- and medium-calibre vascular grafts plays a significant role in graft occlusion. We examined platelet accumulation on the surface of fibrin-coated polyethylene tubing (internal diameter 0.17 cm) during 10 min flow (10 ml/min) at high wall shear rate (764 s-1). Washed platelets labelled with 51Cr were resuspended in Tyrode solution containing albumin, apyrase and red blood cells (hematocrit 40%). When the thrombin that was used to form the fibrin-coated surface was inactivated with FPRCH2Cl before perfusion of the tubes with the platelet: red blood cell suspension, the accumulation of platelets was 59,840 +/- 27,960 platelets per mm2, whereas accumulation on fibrin with residual active thrombin was 316,750 +/- 32,560 platelets per mm2 (n = 4). When the fibrin on the surface was cross-linked by including recombinant factor XIII (rFXIII) in the fibrinogen solution used to prepare the fibrin-coated surface, platelet accumulation, after thrombin neutralization, was reduced by the cross-linking from 46,974 +/- 9702 to 36,818 +/- 7964 platelets per mm2 (n = 12, p < 0.01). Platelet accumulation on tubes coated with D-dimer was ten times less than on tubes coated with D-domain; this finding also supports the observation that cross-linking of fibrin with the formation gamma-gamma dimers reduces platelet accumulation on the fibrin-coated surface. Thrombin-activated platelets themselves were shown to cross-link fibrin when they had adhered to it during perfusion, or in a static system in which thrombin was used to form clots from FXIII-free fibrinogen in the presence of platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Factor XIII↗
PubMed1995

Contrasting effects of thrombin and the thrombin receptor peptide, SFLLRN, on aggregation and release of 14C-serotonin by human platelets pretreated with chymotrypsin or serratia marcescens protease.

Chymotrypsin cleaves glycoprotein Ib (GPIb) on platelets and reduces their responsiveness to thrombin; platelets from patients with the Bernard-Soulier syndrome, which lack GPIb, are also less responsive to thrombin than platelets from normal donors. However, Bernard-Soulier platelets respond normally to the thrombin receptor peptide SFLLRN (13). We compared responses of 14C-serotonin-labeled, chymotrypsin-treated platelets (and control platelets) to thrombin (0.25-2 U/ml) and SFLLRN (5-40 microM). Chymotrypsin treatment strongly inhibited thrombin-induced aggregation and release of 14C-serotonin when concentrations of thrombin of 0.5 U/ml or lower were used, even though these responses of control platelets remained near the maximum. In contrast, there was little difference between the responses of control and chymotrypsin-treated platelets to SFLLRN, even when the responses of control platelets were less than maximal. Thus, chymotrypsin treatment greatly inhibits the response to thrombin of the seven transmembrane domain thrombin receptor cloned by Coughlin's group (1, 2). Since Serratia marcescens protease also hydrolyses GPIb, but has less effect than chymotrypsin on other glycoproteins, we pretreated platelets with several concentrations of S. marcescens protease. Concentrations that abolished aggregation and release of 14C-serotonin in response to thrombin had little effect on these responses to SFLLRN. One interpretation of these findings would be that by cleaving GPIb, both proteases are affecting an interaction that may be important for activation of the cloned receptor by thrombin, but irrelevant to activation of this receptor by SFLLRN.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Role of platelets in thrombosis and hemostasis.

Platelet aggregates, stabilized by fibrin, rapidly form hemostatic plugs when blood vessels are severed or arterial thrombi at sites of vessel injury, such as ruptured atherosclerotic plaques, or regions where blood flow is disturbed, such as at stenoses. These thrombi cause the thromboembolic complications of atherosclerosis: heart attacks, strokes, and peripheral vascular disease. Platelet adhesion to subendothelial components such as collagen activates signalling pathways that lead to thromboxane A2 formation and secretion of platelet granule contents, including ADP. Both these substances cause platelet aggregation, a process in which the integrin, glycoprotein IIb/IIIa, becomes a receptor for fibrinogen, which forms bridges between adjacent platelets. On the surface of stimulated platelets, coagulation is accelerated and thrombin is generated; it is a potent inducer of platelet aggregation and secretion and also causes fibrin to form around the aggregates, stabilizing them. There are receptors on the platelet surface for thrombin, thromboxane A2, collagen, ADP, platelet-activating factor, fibrinogen, von Willebrand factor, and other ligands. Agents that inhibit platelet aggregation and the signalling pathways that are activated by the various aggregating agents are under intensive investigation in many laboratories.

Animals↗
PubMed1994

Effects of chronic administration of ethanol on platelets from rabbits with diet-induced hypercholesterolemia. Unchanged characteristics and responses to ADP but reduction of enhanced thrombin-induced, TxA2-independent platelet responses.

To investigate the effect on platelet function of the interaction between dietary cholesterol and moderate, chronic doses of ethanol, hypercholesterolemia was induced in rabbits by 8 weeks of administration of a chow diet with added (0.25% wt/wt) cholesterol; during the eighth week, a moderate amount of ethanol (6% in drinking water) was given. Blood alcohol levels were not detectable in ethanol-treated rabbits at the time of exsanguination. Ethanol did not affect plasma cholesterol levels or the cholesterol to phospholipid molar ratio in platelets. Platelet membrane fluidity, which decreased with cholesterol feeding, was not altered further by administration of ethanol. The overall fatty acid composition of platelet phospholipids was not affected by either cholesterol feeding or chronic ethanol intake. Responses of washed platelets stimulated with either ADP or thrombin were studied to determine whether ethanol administration modified platelet functions in hypercholesterolemia. Primary ADP-induced aggregation was not affected by cholesterol feeding or chronic ethanol intake, but thrombin-induced aggregation and secretion of [14C]serotonin from prelabeled platelets, which were enhanced by cholesterol feeding, were diminished by administration of ethanol to hypercholesterolemic rabbits. This reduction in thrombin-induced responses was also observed with aspirin-treated platelets, which cannot form thromboxane A2. Thus, chronic short-term administration of a moderate amount of ethanol inhibited the enhanced responses of platelets from rabbits with diet-induced hypercholesterolemia, via a thrombin-induced, thromboxane A2-independent pathway.

Adenosine Diphosphate↗
PubMed1994

Enhanced collagen-induced responses of platelets from rabbits with diet-induced hypercholesterolemia are due to increased sensitivity to TxA2. Response inhibition by chronic ethanol administration in hypercholesterolemia is due to reduced TxA2 formation.

The effects of dietary cholesterol and chronic administration of moderate amounts of ethanol on collagen-induced platelet responses were investigated. Three groups of rabbits were fed the following diets for 8 weeks: a normal chow diet, a cholesterol-enriched (0.25% wt/wt) chow diet, and a cholesterol-enriched chow diet plus 6% ethanol in the drinking water for the final week of the dietary period. Cholesterol feeding enhanced collagen-induced responses-aggregation, secretion of [14C]serotonin from prelabeled platelets, and thromboxane formation--of suspensions of washed platelets, and chronic ethanol treatment significantly reduced these enhanced responses. These effects are mediated by thromboxane A2 (TxA2) rather than ADP. Experiments with collagen-stimulated platelets in which feedback amplification of TxA2 was blocked with the prostaglandin H2/TxA2 receptor blocker BM 13.177 and experiments with aspirin-treated platelets stimulated with the stable TxA2 mimetic U46619 showed that cholesterol feeding enhanced platelet sensitivity to TxA2 rather than formation of TxA2 by platelets that had interacted with collagen. Without BM 13.177 or aspirin, TxA2 increased the amount of TxA2 formed by feedback amplification. In contrast, decreased responsiveness to collagen by platelets from cholesterol-fed rabbits given ethanol was due to inhibition of TxA2 formation rather than reduced sensitivity to TxA2. Platelets from cholesterol-fed rabbits given ethanol did not develop tolerance to the acute inhibitory effects of ethanol. Our results indicate that administration of moderate amounts of ethanol to cholesterol-fed rabbits inhibits enhanced collagen-induced responses of platelets by a TxA2-dependent pathway that involves reduction of TxA2 formation rather than reduction of platelet responses to TxA2.

Alcoholism↗
PubMed1994

Ticlopidine facilitates the deaggregation of human platelets aggregated by thrombin.

Normal human platelets aggregated by thrombin undergo the release reaction and are not readily deaggregated by the combination of inhibitors hirudin, prostaglandin E1 (PGE1) and chymotrypsin. Released adenosine diphosphate (ADP) plays an important role in the stabilization of thrombin-induced human platelet aggregates. Since ticlopidine inhibits the platelet responses to ADP, we studied thrombin-induced aggregation and deaggregation of 14C-serotonin-labeled platelets from 12 patients with cardiovascular disease before and 7 days after the oral administration of ticlopidine, 250 mg b.i.d. Before and after ticlopidine, platelets stimulated with 1 U/ml thrombin aggregated, released about 80-90% 14C-serotonin and did not deaggregate spontaneously within 5 min from stimulation. Before ticlopidine, hirudin (5x the activity of thrombin) and PGE1 (10 mumol/l) plus chymotrypsin (10 U/ml) or plasmin (0.06 U/ml), added at the peak of platelet aggregation, caused slight or no platelet deaggregation. After ticlopidine, the extent of platelet deaggregation caused by the same inhibitors was significantly greater than before ticlopidine. The addition of ADP (10 mumol/l) to platelet suspensions 5 s after thrombin did not prevent the deaggregation of ticlopidine-treated platelets. Thus, ticlopidine facilitates the deaggregation of thrombin-induced human platelet aggregates, most probably because it inhibits the effects of ADP on platelets.

Adenosine Diphosphate↗
PubMed1993

Degranulation of human platelets by the thrombin receptor peptide SFLLRN: comparison with degranulation by thrombin.

A new, simplified method of degranulating human platelets using the thrombin receptor peptide SFLLRN (20 microM) is described; released fibrinogen cannot be converted to fibrin, and the platelets are not exposed to a proteolytic enzyme, as they are when thrombin is used for degranulation. The peptide-degranulated platelets regain their disc shape and are recovered as single platelets which have released approximately 90% of the contents of their dense granules. Their procoagulant activity is greater than that of control platelets, but somewhat less than that of thrombin-degranulated platelets. Without added fibrinogen, the peptide-degranulated platelets aggregate slightly in response to 50 microM SFLLRN, and to collagen, arachidonic acid, the thromboxane A2 mimetic U46619, platelet activating factor, ADP, and the divalent cation ionophore A23187; added fibrinogen enhances aggregation caused by these agonists. Extensive aggregation of peptide-degranulated platelets is caused by thrombin in the absence of added fibrinogen; it may be that the alternative thrombin receptor that is not activated by SFLLRN is responsible for the strong response to thrombin. Aggregation responses to most of the agonists are greater than those observed previously with thrombin-degranulated platelets. By this method, platelets are obtained that have been degranulated in a way that is similar to in vivo degranulation. They are useful for studies of platelet responses without the complicating effects of released granule contents, and for investigation of the characteristics and functions of platelets that have come in contact with release-inducing agents in vivo.

Amino Acid Sequence↗
PubMed1993

Rabbit and rat platelets do not respond to thrombin receptor peptides that activate human platelets.

Human platelets are aggregated and induced to release their granule contents and form thromboxane by peptides as short as 6-amino acid residues (SFLLRN) corresponding to the newly released N-terminus of the thrombin receptor that is cleaved by thrombin. Using washed platelets, we found that these responses to SFLLRN (2 to 6 mumol/L) were enhanced by fibrinogen. However, neither SFLLRN nor SFLLRNPNDKYEPF had any effect on washed rabbit or rat platelets, although they were fully responsive to human thrombin. Concentrations of the peptides as high as 100 mumol/L did not cause the platelets of rabbits or rats to change shape, aggregate, release granule contents, or form thromboxane. SFLLRN did not affect the extent of aggregation induced by adenosine diphosphate (ADP) or a low concentration of thrombin. Pig platelets responded to 50 mumol/L SFLLRN with reversible aggregation, which was enhanced by fibrinogen, but not accompanied by the release of dense granule contents. Guinea pig platelets aggregated and released granule contents in response to 25 or 50 mumol/L of SFLLRN, but responded with only shape change to lower concentrations. Thus, these experiments indicate that rabbit and rat platelets lack a functional response to human thrombin receptor peptides that fully activate the previously described human thrombin receptor, despite a full response of both rabbit and rat platelets to human thrombin, and that pig and guinea pig platelets have incomplete responses to these human thrombin receptor peptides. The results suggest that platelets of rabbits and rats, and perhaps guinea pigs and pigs, respond to thrombin through an alternative receptor that has also been suggested to be present on human platelets.

Amino Acid Sequence↗
PubMed1993

Activation of phospholipase C and protein kinase C has little involvement in ADP-induced primary aggregation of human platelets: effects of diacylglycerols, the diacylglycerols, the diacylglycerol kinase inhibitor R59022, staurosporine and okadaic acid.

The primary phase of ADP-induced aggregation of human platelets does not involve appreciable formation of thromboxane A2 or release of granule contents; lack of formation of inositol trisphosphate has also been noted. Because these responses of platelets to ADP differ so markedly from their responses to other aggregating agents, the roles in ADP-induced aggregation of diacylglycerol, protein kinase C, increases in cytosolic [Ca2+], phosphorylation of pleckstrin (47 kDa) and phosphatases 1 and 2a were investigated. Washed human platelets, prelabelled with [14C]5-hydroxytryptamine and suspended in Tyrode solution (2 mM Ca2+, 1 mM Mg2+), were used for comparisons between the aggregation induced by 2-4 microM ADP, in the presence of fibrinogen, and that induced by 0.05 units/ml thrombin. The diacylglycerol kinase inhibitor 6-(2-[(4-fluorophenyl)phenyl-methylene]-1-piperidinylethyl)-7-meth yl-5H-thiazolo[3,2-a]-pyrimidin-5-one (R59022; 25 microM) had no, or only a slight, enhancing effect on ADP-induced aggregation, but potentiated thrombin-induced responses to a much greater extent. 1,2-Dihexanoyl-sn-glycerol or 1-oleoyl-2-acetyl-sn-glycerol (25 microM) added with or 30-90 s before ADP greatly potentiated aggregation without formation of thromboxane; staurosporine, an inhibitor of protein kinase C, reduced this potentiation. Staurosporine (25 nM) did not inhibit ADP-induced aggregation, although it strongly inhibited thrombin-induced aggregation and release of [14C]5-hydroxytryptamine. All these observations indicate little or no dependence of primary ADP-induced aggregation on the formation of diacylglycerol or on the activation of protein kinase C. At 2-4 microM, ADP did not significantly increase the phosphorylation of pleckstrin (studied with platelets prelabelled with [32P]orthophosphate), but 1,2-dihexanoyl-sn-glycerol- induced phosphorylation of pleckstrin was increased by ADP. Surprisingly, the diacylglycerols strongly inhibited the ADP-induced rise in cytosolic [Ca2+] concurrently with potentiation of ADP-induced aggregation; thus the extent of primary aggregation is independent of the level to which cytosolic [Ca2+] rises. Incubation of platelets with 1,2-dihexanoyl-sn-glycerol or 1-oleoyl-2-acetyl-sn-glycerol for several minutes reversed their potentiating effects on aggregation, and inhibition was observed. Incubation of platelets with okadaic acid, an inhibitor of phosphatases 1 and 2a, inhibited ADP- and thrombin-induced aggregation; although the reason for this effect is unknown, it is unlikely to involve inhibition of phospholipase C, since formation of diacylglycerol appears to have little involvement in the primary phase of ADP-induced aggregation.

Adenosine Diphosphate↗
PubMed1993

Role of platelets in hepatic allograft preservation injury in the rat.

Cold preservation of liver allografts injuries hepatic sinusoidal lining cells. This injury is exacerbated on reperfusion, in part because of adhesion of leukocytes. Platelets also adhere to activated endothelial surfaces. In this study we examined the role of platelets in preservation injury. Our specific aim was to determine whether the degree of platelet adhesion on reperfusion of preserved rat livers was related to duration of cold or warm ischemia and whether platelet adhesion resulted in injury to allografts. We also examined the effect of prior activation of platelets on adhesion and injury. Rat livers were preserved at 1 degree C for different time periods in University of Wisconsin solution and then reperfused for 3 hr on the isolated perfused rat liver system with Krebs-Henseleit solution to which unactivated isolated rat platelets were added. Other livers were rewarmed before reperfusion or reperfused with activated platelets. Platelets were lost from the circulation in all studies; the percentage reduction of circulating platelets was dependent on the length of preservation. The initial platelet concentration did not affect the rate of reduction of platelets in the circuit. Rewarming before reperfusion increased platelet adherence, and prior activation also increased adherence. With electron microscopy we determined that platelets adhered in small aggregates to endothelial cells or endothelial cell remnants. Adherent platelets appeared more activated and contained fewer granules than did unperfused platelets. Liver injury as measured by release of transaminases into perfusate was worsened by longer periods of cold preservation and by addition of rewarming to the protocol. The presence of platelets under these circumstances aggravated injury. Prior activation of platelets also increased the extent of injury. These studies show that platelets have an important role in cold preservation-reperfusion injury.

Alanine Transaminase↗
PubMed1992

Unexpected effects of aurin tricarboxylic acid on human platelets.

Aurin tricarboxylic acid (ATA) is a potent inhibitor of ristocetin-mediated platelet agglutination and of shear-induced, von Willebrand factor (vWf)-mediated platelet aggregation, probably via inhibition of vWf interaction with glycoprotein Ib (GPIb). We examined the effects of ATA (both the sodium salt and a solution of ATA in ethanol) on platelet functions in citrated plasma (PRP) and in suspensions of washed platelets in Tyrode-albumin solution (contains 2 mM Ca2+). ATA (42-211 micrograms/ml) blocked aggregation and release of granule contents induced by thrombin (0.15 U/ml in PRP; 0.03 U/ml in platelet suspension). Responses to higher concentrations of thrombin were not inhibited. ATA also prolonged thrombin-induced clotting of fibrinogen. Since ATA had no effect on fibrinogen-induced responses of chymotrypsin-treated platelets, ATA probably acts on thrombin rather than on fibrinogen. In PRP and platelet suspensions, ATA (acid form 106 micrograms/ml; sodium salt 122 micrograms/ml) had little effect on ADP-induced platelet aggregation. The sodium salt of ATA (61-122 micrograms/ml) enhanced collagen-induced aggregation and release by platelets in citrated plasma and by washed platelets; the enhancement was extensively inhibited by aspirin. With platelet suspensions, ATA significantly enhanced aggregation and release caused by low concentrations of sodium arachidonate (15-50 microM); aggregation and release caused by higher concentrations of arachidonate were somewhat inhibited by ATA. Arachidonate-induced aggregation and release were also enhanced by ATA in PRP. ATA enhanced aggregation and release induced by the calcium ionophore A23187; aspirin had little effect on the enhancement.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1992

Lack of stability of aggregates after thrombin-induced reaggregation of thrombin-degranulated platelets.

The stability of platelet aggregates is influenced by the extent of the release of granule contents; if release is extensive and aggregation is prolonged, deaggregation is difficult to achieve. The relative importance of the contributions of released substances to aggregate stability are not known, although stable thrombin-induced aggregates form in platelet-rich plasma from patients with barely detectable plasma or platelet fibrinogen, and ADP stabilizes thrombin-induced aggregates of platelets from patients with delta storage pool deficiency which otherwise deaggregate more readily than normal platelets. We degranulated platelets with thrombin (0.9 U/ml caused greater than 90% loss of delta and alpha granule contents) and recovered them as individual platelets in fresh medium. The degranulated platelets were reaggregated by thrombin (2 U/ml). To prevent continuing effects of thrombin, FPRCH2Cl was added when thrombin-induced aggregation of thrombin-degranulated platelets reached its maximum. EDTA (5 mM) or EGTA (5 mM) added at maximum aggregation did not deaggregate these platelets, indicating that the stability of these aggregates does not depend on Ca2+ in the medium. Whereas with control platelets a combination of PGE1 (10 microM) and chymotrypsin (10 U/ml) was required for deaggregation, with thrombin-degranulated platelets either PGE1 or chymotrypsin alone caused extensive deaggregation. The rate and extent of deaggregation of thrombin-degranulated platelets by a combination of PGE1 and chymotrypsin was greater than with control platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1992

Acute in vitro effects of ethanol on responses of platelets from cholesterol-fed and Watanabe heritable hyperlipidemic rabbits.

The effects of ethanol on platelets from rabbits with two different types of hypercholesterolemia, diet-induced and genetically determined, were investigated. There were no differences between the hypercholesterolemic groups and their controls in the extent of (primary) ADP-induced aggregation of washed platelets, and this aggregation was not inhibited by ethanol. Platelets from cholesterol-fed rabbits were more sensitive to aggregation and secretion induced by collagen, whereas platelets from Watanabe heritable hyperlipidemic (WHHL) rabbits were less sensitive. Ethanol inhibited collagen-induced responses of platelets from both hypercholesterolemic groups, but the extent of inhibition of aggregation was not different compared with controls. Because ethanol did not affect U46619-induced responses of aspirin-treated platelets, ethanol does not inhibit aggregation and secretion stimulated by collagen via an effect on thromboxane A2 (TxA2)-induced responses. Platelets from cholesterol-fed rabbits were more sensitive to thrombin even when TxA2 formation was blocked by aspirin, and inhibition of aggregation by ethanol was less in cholesterol-fed rabbits than in controls. However, neither the extent of thrombin-induced responses nor the inhibitory effect of ethanol was different in platelets from WHHL rabbits compared with controls. Thus, different etiologies of hypercholesterolemia produce different changes in platelet function, and ethanol has different effects on the platelets from cholesterol-fed rabbits compared with the platelets from WHHL rabbits. The inhibitory effect of ethanol on the thrombin-induced aggregation of platelets from cholesterol-fed rabbits is attenuated compared with controls, and this finding contrasts with the reported greater inhibitory effect of ethanol on platelets enriched with saturated fats.

Adenosine Diphosphate↗
PubMed1991

Characteristics of thrombin-degranulated human platelets: development of a method that does not use proteolytic enzymes for deaggregation.

A method has been developed for preparing suspensions of washed human platelets that have lost as much as 90% of their dense granule and alpha granule contents as a result of stimulation by thrombin (0.9 U/ml for 3 min at 37 degrees C), and recovering the platelets without using a proteolytic enzyme. Glycyl-L-prolyl-L-arginyl-L-proline (GPRP) was used to prevent polymerization of released fibrinogen and arginyl-glycyl-aspartyl-serine (RGDS) to block the interaction of released fibrinogen, vWf or fibronectin with the glycoprotein IIb/IIIa complex. The thrombin used to degranulate the platelets was neutralized with D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (FPRCH2Cl) and prostaglandin E1 was added to return the platelets towards a disc shape. The degranulated platelets aggregated in response to ADP, platelet activating factor, arachidonate and the thromboxane A2 mimetic, U46619 in the presence of added fibrinogen; the platelets changed shape but did not aggregate in response to collagen. Thrombin and the calcium ionophore, A23187, caused aggregation without added fibrinogen. Synergism between pairs of aggregating agents at low concentrations was observed. Little TXB2 was formed when the platelets were reaggregated by thrombin. RGDS and F(ab')2 fragments of an antibody to fibrinogen inhibited reaggregation induced by thrombin and A23187 indicating that small amounts of fibrinogen at the platelet surface may support aggregation by strong agonists. Adherence of thrombin-degranulated platelets to a collagen-coated surface was less than for controls, but spreading was more extensive. Electron-microscopic immunogold cytochemistry with anti-human fibrinogen IgG showed numerous gold particles in platelet vacuoles.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗