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S J Shattil

Publications and source records attributed to S J Shattil.

At least 109 records · Page 6Linked to original sources
PubMed1986

Desensitization of epinephrine-initiated platelet aggregation does not alter binding to the alpha 2-adrenergic receptor or receptor coupling to adenylate cyclase.

Several investigators have shown that incubating unstirred platelets with epinephrine blunts subsequent aggregation when the platelets are stirred. Using aspirin-treated platelets, we further characterized this desensitization of alpha 2-adrenergic receptor-initiated aggregation. Desensitization occurred with a t1/2 of 3-6 min and was maximal at 20-30 min, at which time the initial rate of aggregation and its maximal extent were about half that of control platelets. When we preincubated platelets with epinephrine, and then added phentolamine to block the alpha 2-receptors, ADP-initiated aggregation occurred normally. Thus, the desensitization of epinephrine-initiated aggregation was not associated with a generalized impairment of aggregation. At concentrations too low to initiate aggregation, epinephrine is known to potentiate aggregation initiated by other agents. Clonidine also acts at alpha 2-receptors to potentiate aggregation initiated by other agents, but it does not initiate aggregation by itself. Preincubating clonidine with platelets for 30 min abolished its potentiating effect on ADP-initiated aggregation. Thus, the ability of alpha 2-receptors to both potentiate and initiate aggregation desensitizes after a brief preincubation with agonist. We performed several types of experiments to investigate the mechanism of this desensitization. Platelet alpha 2-receptors are coupled to an inhibition of adenylate cyclase. We found, however, that alpha 2-mediated inhibition of prostaglandin E1-stimulated cAMP accumulation occurred normally in desensitized platelets. Similarly, epinephrine inhibited basal adenylate cyclase activity normally in membranes prepared from desensitized platelets. In membranes prepared from desensitized platelets, epinephrine competed normally for [3H]rauwolscine binding, and this competition was modulated normally by guanine nucleotides. Thus, the properties of the alpha 2-receptors, as measured in radioligand binding experiments, were unchanged by densensitization. In conclusion, desensitization of alpha 2-adrenergic receptor-mediated aggregation occurs without change in the alpha 2-adrenergic receptors or in their coupling to an inhibition of adenylate cyclase.

1-Methyl-3-isobutylxanthine↗
PubMed1986

Expression of fibrinogen receptors during activation and subsequent desensitization of human platelets by epinephrine.

Epinephrine causes platelet aggregation and secretion by interacting with alpha 2-adrenergic receptors on the platelet surface. Platelet aggregation requires the binding of fibrinogen to a specific receptor on the membrane glycoprotein IIb-IIIa complex. Although the IIb-IIIa complex is identifiable on the surface of resting platelets, the fibrinogen receptor is expressed only after platelet activation. The current studies were designed to examine the effect of occupancy of platelet alpha 2-adrenergic receptors by epinephrine on the expression of fibrinogen receptors and on the aggregation of platelets. The ability of epinephrine to induce the expression of fibrinogen receptors was studied under two different conditions: acute stimulation (less than 1 min) and prolonged stimulation (50 to 90 min), the latter of which is associated with a reduction or "desensitization" of the platelet aggregation response. Expression of the fibrinogen receptor was monitored with 125I-fibrinogen as well as with 125I-PAC-1 (PAC-1), a monoclonal antibody that binds to the glycoprotein IIb-IIIa complex only after platelets are activated. Epinephrine caused an immediate increase in PAC-1 and fibrinogen binding that was dependent on occupancy of the alpha 2-receptor by epinephrine and on the presence of extracellular free Ca (KCa = 30 mumol/L). By itself, 1 mmol/L Mg was unable to support induction of the fibrinogen receptor by epinephrine. However, it did decrease the Ca requirement by about two orders of magnitude. Prolonged stimulation of unstirred platelets by epinephrine led to a 70% decrease in the aggregation response when the platelets were subsequently stirred. Despite their decreased aggregation response, desensitized platelets bound PAC-1 and fibrinogen normally, indicating that the loss of aggregation was not due simply to a decrease in fibrinogen receptor expression. Although desensitization was not affected by pretreatment of the platelets with aspirin, it was partially prevented when extracellular Ca was chelated by EDTA during the long incubation with epinephrine. These studies demonstrate that once platelet alpha 2-adrenergic receptors are occupied by epinephrine, extracellular Ca is involved in initiating the aggregation response by supporting the induction of the fibrinogen receptor and the binding of fibrinogen. Furthermore. Ca-dependent reactions subsequent to fibrinogen binding may be necessary for maximal platelet aggregation and are impaired when platelets become desensitized to epinephrine.

Antibodies, Monoclonal↗
PubMed1985

Changes in the platelet membrane glycoprotein IIb.IIIa complex during platelet activation.

Platelet activation is accompanied by the appearance on the platelet surface of approximately 45,000 receptor sites for fibrinogen. The binding of fibrinogen to these receptors is required for platelet aggregation. Although it is established that the fibrinogen receptor is localized to a heterodimer complex of the membrane glycoproteins, IIb and IIIa, little is known about the changes in this complex during platelet activation that result in the expression of the receptor. In the present studies, we have developed and characterized a murine monoclonal anti-platelet antibody, designated PAC-1, that binds to activated platelets, but not to unstimulated platelets. PAC-1 is a pentameric IgM that binds to agonist-stimulated platelets with an apparent Kd of 5 nM. Binding to platelets is dependent on extracellular Ca2+ (KCa = 0.4 microM) but is not dependent on platelet secretion. Platelets stimulated with ADP or epinephrine bind 10,000-15,000 125I-PAC-1 molecules/platelet while platelets stimulated with thrombin bind 20,000-25,000 molecules/platelet. Several lines of evidence indicate that PAC-1 is specific for the glycoprotein IIb.IIIa complex. First, PAC-1 binds specifically to the IIb.IIIa complex on Western blots. Second, PAC-1 does not bind to thrombasthenic platelets or to platelets preincubated with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid at 37 degrees C, both of which lack the intact IIb.IIIa complex. Third, PAC-1 competitively inhibits the binding of 125I-A2A9, and IgG monoclonal antibody that is specific for the IIb.IIIa complex. Fourth, the antibody inhibits fibrinogen-mediated platelet aggregation. These data demonstrate that PAC-1 recognizes an epitope on the IIb.IIIa complex that is located near the platelet fibrinogen receptor. Platelet activation appears to cause a Ca2+-dependent change involving the glycoprotein IIb.IIIa complex that exposes the fibrinogen receptor and, at the same time, the epitope for PAC-1.

Antibodies, Monoclonal↗
PubMed1985

Effect of calcium on the stability of the platelet membrane glycoprotein IIb-IIIa complex.

Platelet membrane glycoproteins IIb and IIIa form a Ca2+-dependent heterodimer complex that contains binding sites for fibrinogen, von Willebrand factor, and fibronectin following platelet stimulation. We have studied the effect of Ca2+ on the stability of the IIb-IIIa complex using a IIb-IIIa complex-specific monoclonal antibody A2A9 to detect the presence of the complexes. Soluble IIb and IIIa interacted with A2A9-Sepharose only in the presence of Ca2+ with 50% IIb-IIIa binding requiring 0.4 microM Ca2+. In contrast, at 25 degrees C 125I-A2A9 binding to intact unstimulated platelets suspended in buffers containing EDTA or ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid was independent of the presence of Ca2+. However, the effect of Ca2+ chelators on 125I-A2A9 binding varied with temperature. At 37 degrees C, 125I-A2A9 binding to intact platelets became Ca2+-dependent with 50% binding requiring 0.4 microM Ca2+. This effect of temperature was not due to a change in platelet membrane fluidity because enrichment or depletion of platelet membrane cholesterol did not influence antibody binding. But, 125I-A2A9 binding to intact platelets at 25 degrees C did become Ca2+-dependent when the pH was increased above 7.4. Thus, at 1 nM Ca2+ and 25 degrees C, 50% antibody binding occurred at pH 9.0. Our studies demonstrate that Ca2+-dependent IIb-IIIa complexes are present on unstimulated platelets and that the Ca2+ binding sites responsible for the stability of these complexes are located on the external platelet surface. Our experiments also suggest that changes in platelet cytosolic Ca2+ do not regulate the formation of IIb-IIIa complexes.

Animals↗
PubMed1985

The interaction of extracellular calcium with the platelet membrane glycoprotein IIb-IIIa complex.

Platelet aggregation requires the presence of extracellular Ca2+ and fibrinogen. When platelets are activated, fibrinogen receptors become expressed on the cell surface. These receptors are composed of a heterodimer complex of two integral membrane glycoproteins, IIb and IIIa. The requirement for Ca2+ in platelet aggregation can be explained in part by the interaction of Ca2+ with this complex. Conceptually, this interaction can be divided into three parts. First, Ca2+ holds the membrane IIb-IIIa complex together. Half-maximal dissociation of the complex occurs at 37 degrees in the presence of 0.4 uM extracellular Ca2. Mg2+ ions are unable to serve as a substitute for Ca2+. Second, studies with a complex-specific monoclonal antibody that binds only to activated platelets indicate that Ca2+ also is required for the agonist-induced conversion of the IIb-IIIa complex into a functional fibrinogen receptor. The KCa for receptor expression is also 0.4 uM, and Mg2+ is unable to substitute for the Ca2+. Third, Ca2+ is required for the actual binding of fibrinogen to its receptor. In this case, the KCa is 10-100 uM, and Mg2+ is an effective substitute for Ca2+. Thus, Ca2+ holds the IIb-IIIa complex together, is involved in fibrinogen receptor expression and supports fibrinogen binding. These multiple interactions of extracellular Ca2+ with glycoproteins IIb and IIIa help to explain the known requirement for Ca2+ in platelet aggregation.

Blood Platelets↗
PubMed1985

Biochemical and functional consequences of dissociation of the platelet membrane glycoprotein IIb-IIIa complex.

The platelet membrane glycoproteins, IIb and IIIa, form a Ca2+-dependent heterodimer complex that functions as the fibrinogen receptor in activated platelets to mediate platelet aggregation. Little is known about factors that affect the IIb-IIIa complex within the platelet membrane. It has been observed that platelets incubated with ethylene glycol tetra-acetic acid (EGTA) at 37 degrees C are unable to aggregate or to bind monoclonal antibodies specific for the IIb-IIIa complex. To determine whether this is due to a dissociation of IIb from IIIa, we developed a method for quantitating the complex on nondenaturing, polyacrylamide gradient gels. Platelets were surface-labeled with 125I and then solubilized and electrophoresed in 0.2% Triton and 10 mmol/L CHAPS. Under these conditions and in the presence of 1 mmol/L Ca2+, glycoproteins IIb and IIIa migrated on the gels as a discrete band at Rf = 0.33. Protein that was eluted from this band bound to an immunoaffinity column specific for the IIb-IIIa complex. In contrast, when the IIb-IIIa complex was solubilized and then dissociated with EGTA, the discrete band at Rf = 0.33 was no longer present, and IIb and IIIa were now found in a broad band at Rf = 0.45 to 0.50. To study IIb and IIIa within the surface membrane, the 125I-labeled platelets were first incubated with 0.5 mmol/L EGTA (1 nmol/L free Ca2+) at 22 degrees C and then solubilized in the absence of EGTA. The IIb and IIIa from these platelets migrated at Rf = 0.33, indicating the presence of the intact IIb-IIIa complex. In contrast, when the platelets were incubated at 37 degrees C for one hour with the EGTA, the discrete band at Rf = 0.33 representing the IIb-IIIa complex gradually disappeared. This phenomenon could not be reversed by adding Ca2+ back to the platelets before solubilization and electrophoresis. This loss of the IIb-IIIa complex from intact platelets was accompanied by (a) a progressive and irreversible decrease in adenosine diphosphate (ADP)-induced platelet aggregation and (b) decreased binding of a complex-dependent monoclonal antibody to the platelets. These studies demonstrate that when platelets are exposed to low Ca2+ at 37 degrees C, the IIb-IIIa heterodimer complexes in their surface membranes are irreversibly disrupted. Because intact IIb-IIIa complexes are required for platelet aggregation, the loss of these complexes may account for the failure of these platelets to aggregate in response to ADP.

Blood Platelets↗
PubMed1984

Diagnosis and treatment of recurrent venous thromboembolism.

The diagnosis of recurrent venous thromboembolism includes an evaluation of the patient for inherited or acquired disorders, associated with an increased risk of thromboembolism. Treatment depends, in part, on the underlying disease and, in part, on an adequate understanding of the principles of anticoagulant therapy.

Anticoagulants↗
PubMed1984

Identification and function of the high affinity binding sites for Ca2+ on the surface of platelets.

Extracellular Ca2+ is required for platelet aggregation and secretion in response to ADP or epinephrine. Recently, we reported that the platelet surface contains two classes of high affinity binding sites for extracellular Ca2+. To identify these sites and clarify their role in platelet function, we have now (a) studied platelets congenitally deficient in surface membrane glycoproteins and (b) examined the effect of removing surface-bound Ca2+ on platelet responses to ADP and epinephrine. Unstimulated normal platelets contained 86,000 Ca2+-binding sites/platelet with a dissociation constant (Kd) of 9 nM and 389,000 sites with a Kd of 400 nM. In contrast, thrombasthenic platelets, which lack glycoproteins IIb and IIIa, exhibited a 92% reduction in the number of higher affinity Ca2+-binding sites and a 63% reduction in the number of lower affinity sites. Bernard-Soulier platelets, which lack glycoprotein Ib, were not deficient in Ca2+-binding sites. After stimulation with ADP, both normal and thrombasthenic platelets developed approximately 138,000 new Ca2+-binding sites/platelet (Kd = 400 nM), while the larger Bernard-Soulier platelets developed 216,000 new sites. These data suggest that IIb and IIIa represent the major Ca2+-binding glycoproteins on unstimulated platelets, while neither these glycoproteins nor Ib represent the new Ca2+-binding sites on stimulated platelets. Removal of Ca2+ from the platelet surface inhibited platelet function. Despite the presence of 1 mM Mg2+, ADP- and epinephrine-induced aggregation and [14C]serotonin release were markedly decreased at free Ca2+ concentrations less than 7 nM, a value similar to the Kd of the higher affinity Ca2+-binding sites. Moreover, gadolinium, a lanthanide that competed for these Ca2+-binding sites, also inhibited aggregation and serotonin release. These studies demonstrate, therefore, that the binding of extracellular Ca2+ to glycoproteins IIb/IIIa on unstimulated platelets or to additional membrane proteins on stimulated platelets is necessary for maximal platelet responses to ADP and epinephrine. Thus, the requirement for extracellular Ca2+ during platelet activation by these agonists may actually represent a requirement for surface-bound Ca2+.

Adenosine Diphosphate↗
PubMed1982

Changes in surface-bound and exchangeable calcium during platelet activation.

Using 45CaCl2, we measured the effect of ADP and epinephrine on exchangeable Ca2+ pools in gel-filtered platelets. Unstimulated platelets contained 1) a slowly exchangeable pool, representing 54% of the exchangeable Ca2+, that was unaffected by ethylene glycol bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid and was, therefore, presumably intracellular; and 2) a rapidly exchangeable pool, representing 46% of the Ca2+, that appeared to be surface-bound because it was removable by ethylene glycol bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid or LaCl3. Equilibrium studies of this surface-bound Ca2+ revealed two classes of saturable binding sites, one class of 57,000 sites with a high affinity (KD = 5 x 10(-9) M) and another of 460,000 sites with a lower affinity for Ca2+ (KD = 3 x 10(-7) M). Two changes were observed with ADP and epinephrine. First, the amount of surface-bound Ca2+ increased by up to 50%. In the case of ADP, this included an increase in the number of lower affinity Ca2+ binding sites by 28%. Epinephrine increased the number of lower affinity sites by 18% (p less than 0.001). Second, while not increasing the amount of intracellular Ca2+, both agonists increased by 40% the rate of Ca2+ exchange into the intracellular pool. These effects were rapid (less than 1 min), dependent on agonist concentration, and inhibited by prostaglandin I2 or by specific antagonists of ADP or epinephrine binding. Thus, we find no evidence for a net influx of Ca2+ into platelets stimulated by ADP or epinephrine. However, both agonists increase the amount of Ca2+ bound to the platelet surface membrane. This bound Ca2+ may participate in several membrane reactions involved in platelet activation.

Adenosine Diphosphate↗
PubMed1982

Interaction of verapamil with human platelet alpha-adrenergic receptors.

Platelet aggregation induced by epinephrine is an alpha-adrenergic event and is inhibited in vitro by the calcium-channel blocker, verapamil. We wondered whether this inhibition might be mediated by an interaction of verapamil with platelet alpha-adrenergic receptors. Verapamil inhibited the specific binding of [3H]dihydroergocryptine ([3H]DHE), a nonselective alpha-adrenergic antagonist, to intact platelets in a concentration-dependent and competitive manner. At 10 microM verapamil caused a fivefold decrease in the affinity of binding of [3H]DHE to platelet alpha-adrenergic receptors (P less than 0.001) with no change in the number of receptors per platelet. The inhibition of binding was not reversed by excess calcium. Verapamil also inhibited the binding of a selective alpha 2-adrenergic antagonist, [3H]yohimbine, to intact platelets, and inhibited the binding of an alpha 2-partial agonist, [3H]clonidine, to platelet membranes. Moreover there was a strong correlation between verapamil's effect on [3H]DHE binding and its effect on epinephrine-induced aggregation (r = 0.98). Although verapamil is commonly used as an inhibitor of calcium flux across cell membranes, the present studies demonstrate that verapamil also inhibits epinephrine-induced aggregation at the level of the platelet alpha-adrenergic receptor.

Binding, Competitive↗
PubMed1982

The effect of pharmacologic inhibition of phospholipid methylation on human platelet function.

Human platelets are capable of synthesizing their major membrane phospholipid, phosphatidylcholine, by a methylation pathway. This involves the sequential transfer of methyl groups from S-adenosyl-L-methionine (AdoMet) to phosphatidylethanolamine, and in the process, AdoMet is converted to S-adenosylhomocysteine (AdoHcy). The activity of this methylation pathway is decreased upon stimulation of platelets by various agonists. We inhibited methylation reactions pharmacologically to see whether this inhibition plays any role in the process of platelet activation. Two inhibitors of AdoHcy hydrolase, 3-deaza-adenosine and 3-deaza-(+/-)aristeromycin (500 microM each), were effective in increasing platelets levels of AdoHcy and preventing turnover of AdoMet. Also, these compounds were equipotent in inhibiting platelet phospholipid methylation. However, while 3-deaza-adenosine potentiated platelet aggregation and 14C-serotonin release induced by epinephrine or adenosine diphosphate (ADP) (p less than 0.01), 3-deaza-aristeromycin had no such effect. Neither compound affected platelet responses to thrombin or collagen. Inhibition of methylation reactions was not the only biochemical effect of 3-deaza-adenosine since it also blunted significantly the elevation of platelet cyclic adenosine monophosphate (AMP) levels induced by prostaglandin E1 (p less than 0.02). Therefore, these studies demonstrate that inhibition of platelet phospholipid methylation, per se, has no discernable effect on the function of human platelets. The methylation pathway, though active in platelets, does not appear to be primarily involved in membrane events responsible for platelet activation.

Adenosine↗
PubMed1981

Platelets and their membranes in hemostasis: physiology and pathophysiology.

Platelets form a plug and promote thrombin generation at sites of vascular injury. These processes are initiated by interaction of the platelet plasma membrane with various substances within or accumulating at the injured vessel. Thus, platelet adhesion to exposed subendothelium requires the binding of von Willebrand factor to platelets. Agonists such as thrombin bind to membrane receptors, thereby stimulating the binding of fibrinogen to platelets and resulting in the aggregation of platelets onto those already adherent to the vessel wall. Agonists also stimulate transfer of membrane=bound calcium into the cytoplasm. This triggers the secretion of granule substances and results in the recruitment of additional platelets to the hemostatic plug. Concomitant with secretion, the platelet surface supports several reactions leading to thrombin generation. Thus, hemostasis requires a series of coordinated responses involving platelet membranes. A defect in any of these responses can lead to a bleeding diathesis.

Blood Platelet Disorders↗
PubMed1981

Inhibition of platelet phospholipid methylation during platelet secretion.

A pathway for the synthesis of membrane phosphatidylcholine involving the N-methylation of phosphatidylethanolamine has been detected in several types of mammalian cells. Furthermore, it has been implicated in the coupling of agonist binding to cell response. We examined whether human platelets exhibit this synthetic pathway and whether platelet agonists influence its activity. When washed platelets were incubated with 0.15 microM L-[methyl-3H]methionine at 37 degrees C, they incorporated methyl-3H into their phospholipids linearly at the rate of 1 pmole/10(9) platelets/hr. When incubated with 20 microM radiolabeled methionine, they incorporated about 15 pmole/10(9) platelets/hr. The radioactivity was found predominantly in phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine, and phosphatidylcholine. Thrombin caused an immediate (within 15 sec) and sustained (up to 30 min) decrease in the rate and extent of N-methylation of platelet phospholipids. This was accounted for by a decrease in synthesis of methylated phospholipids rather than an increase in their degradation. This thrombin effect correlated with serotonin release and could be dissociated from platelet aggregation and prostaglandin synthesis. Thrombin also decreased the synthesis of phosphatidylcholine when choline was used as the radiolabeled substrate. Other agonists such as epinephrine, adenosine diphosphate (ADP), or A23187 also decreased phospholipid methylation under conditions in which they stimulated serotonin release. These data demonstrate that platelets are capable of synthesizing phosphatidylcholine from phosphatidylethanolamine by N-methylation and that agonists perturb this pathway as they induce platelet secretion. The precise role of phospholipid methylation in either resting or stimulated platelets remains to be established.

Adenosine Diphosphate↗
PubMed1980

Carbenicillin and penicillin G inhibit platelet function in vitro by impairing the interaction of agonists with the platelet surface.

Carbenicillin or penicillin G administered in large doses can cause a bleeding diathesis as a result of platelet dysfunction. These antibiotics also inhibit platelet aggregation in vitro, although several-fold larger concentrations of drug are required to demonstrate this effect. We wondered whether these antibiotics might impair platelet function by interfering with the initial step of platelet activation: the binding of agonists to their specific receptors on the platelet surface.Platelet aggregation and [(14)C]serotonin release induced by epinephrine were competitively inhibited by carbenicillin and penicillin G in vitro. At antibiotic concentrations that inhibited platelet function by more than 80%, the affinity of platelet alpha-adrenergic receptors for the alpha-adrenergic antagonist, [(3)H]dihydroergocryptine, and for epinephrine was reduced twofold by carbenicillin and sixfold by penicillin G (P < 0.01). Platelet aggregation and [(14)C]serotonin release stimulated by ADP were also competitively inhibited by these antibiotics. In addition, carbenicillin reduced the incorporation of an ADP affinity label, 5'-p-fluorosulfonylbenzoyl [(3)H]adenosine, into its binding protein in platelet membranes. Moreover, both carbenicillin and penicillin G impaired the interaction of von Willebrand factor with platelets as evidenced by their inhibition of the agglutination of formalin-fixed platelets by ristocetin, snake venom, or bovine factor VIII. These studies demonstrate that carbenicillin and penicillin G inhibit platelet function in vitro by impairing the interaction of several agonists with their specific receptors on the platelet surface membrane. If this were mechanism operative in vivo, it could account for the hemorrhagic as well as the potential antithrombotic effects of these antibiotics.

Adenosine Diphosphate↗