Particulate and vapor phase constituents of cigarette mainstream smoke and risk of myocardial infarction.

On pharmacological and compositional grounds, cigarette mainstream smoke (MS) aerosol can be broadly categorized as consisting of the following constituents: carbon monoxide, other vapor phase components, particulate matter ('tar') and nicotine. The relative risk of coronary artery disease for smoking 20 cigarettes per day has been estimated by meta-analysis of five large prospective epidemiology studies to be 1.78. These four major smoke components are simultaneously delivered to the active smoker as a complex aerosol composed of several billion semi-liquid particles per cm(3) within the mixture of combustion gases. Fractional attribution of the 78% increase in reported risk to a given constituent is problematic because of the complex mixture. However, a significant literature exists which suggests that some general statements regarding smoke constituent-associated risks for development or exacerbation of myocardial infarction are supportable. First, the atherogenic potential of MS is associated with the particulate and vapor phases and not with CO. Nicotine might exert an indirect effect on atherosclerotic plaque development by increasing shear forces in main conduction arteries. Similarly, the thrombogenic potential is associated primarily with the particulate and vapor phases and also possibly with nicotine via platelet aggregation. Vasoconstriction probably results from the actions of nicotine and hypoxia from carbon monoxide. Finally, the arrhythmia-inducing potential may result from catecholamine release following nicotine exposure, with a questionable contribution from carbon monoxide.

Urinary thromboxane, prostacyclin, cortisol, and 8-hydroxy-2'-deoxyguanosine in nonsmokers exposed and not exposed to environmental tobacco smoke.

This study tested the hypotheses that (1) increased platelet aggregation, as measured by 2,3-dinor-thromboxane B(2) (Tx-M) and 2,3-dinor-6-keto-prostaglandin F(1alpha) (PGI-M), and (2) increased oxidative stress, as measured by 8-Hydroxy-2'-deoxyguanosine (8-OHdG), would occur in ETS-exposed nonsmokers as compared with non-ETS-exposed nonsmokers. The concentrations of the stable urinary metabolites of thromboxane (Tx-M) and prostacyclin (PGI-M), cortisol and 8-OHdG were measured in a 24-h urine sample from 3 groups of subjects: 21 nonsmokers with minimal (15 min or less per day) ETS exposure (termed non-ETS-exposed), 22 nonsmokers with at least 5 h per day of ETS exposure (termed ETS-exposed), and 20 cigarette smokers who served as a positive control group. The self-reported levels of ETS exposure were verified by personal air monitors. As compared with either group of nonsmokers, cigarette smokers excreted significantly more urinary Tx-M. Non-ETS-exposed nonsmokers showed a statistically significantly higher level of urinary Tx-M over that seen in nonsmokers with considerably more ETS exposure. Urinary concentrations of PGI-M were marginally higher in the smokers and did not differ between the nonsmoker groups. Nonsmokers exposed to at least five h of ETS per day did not have significantly higher excretion of 8-OHdG than non-ETS-exposed nonsmokers. The results from this study suggest that platelet aggregation, as measured by the thromboxane metabolite Tx-M and prostacyclin metabolite PGI-M, is not associated with ETS exposure. Therefore, platelet aggregation is not a plausible or quantitatively consistent mechanism to explain the nonlinear dose-response hypothesis of cardiovascular disease and ETS exposure.

Splenic clearance mechanisms of rehydrated, lyophilized platelets.

A variety of platelet substitutes (e.g., rehydrated, lyophilized (RL) platelets, thromboerythrocytes, plateletsomes, infusible platelet membranes, synthocytes, fibrinogen-coated microcapules) are potentially useful as hemostatic agents in transfusion medicine. However, as "foreign" particles, platelet substitutes interact to varying extents with elements of the reticulo-endothelial system for clearance, reducing hemostatic efficacy. Experiments were performed to better understand the interaction of RL platelets with elements of the innate and acquired immune systems. The infusion of heterologous RL platelets into rats resulted in rapid clearance from the free circulation with half-life values of minutes. The clearance of RL platelets was inhibited when macrophages were rendered apoptotic with gadolinium. Transmission EM analysis of splenic tissue after infusion of lyophilized cells, as well as in vitro mixing studies with splenic macrophages and RL platelets, indicated that macrophage-mediated phagocytosis mechanisms were operant in RL platelet clearance by the reticulo-endothelial system. Studies with IV IgG, as a competitive inhibitor of the macrophage Fc receptor, provides evidence that RL platelet destruction is in part mediated by platelet surface bound IgG. This hypothesis was further supported by the finding that RL platelets react with IgG class antibodies that are pre-existing in naïve animals. These studies provide a rational basis for prolonging the circulation time of RL platelets and other platelet substitutes.

Role of nuclear factor-kappa B (NF-kappa B) in inflammation, periodontitis, and atherogenesis.

Atherosclerosis, the major cause of death and disability in the United States, is a chronic disease with inflammatory components. The first objective of this review is to explain how activation of NF-kappa B contributes to atherosclerosis. The second objective is to describe a potential link between inflammation, activation of NF-kappa B, and periodontitis. The nuclear transcription factor NF-kappa B controls the expression of many genes linked to atherogenesis including those involved with inflammation. We hypothesize that one unifying mechanism in this complex disease is the activation of NF-kappa B. The mechanism(s) that activates NF-kappa B in atherogenesis is unknown and the effect of inhibiting NF-kappa B activation on atherogenesis is untested. Periodontal disease has now been established as a risk factor for atherosclerosis and its thrombotic complications. It is unknown if periodontal disease contributes to the initiation or progression of atherosclerosis. We hypothesize that the chronic and intense inflammatory response accompanying periodontal disease produces an excess burden of circulating mediators of inflammation that initiate or exacerbate the inflammatory components of atherogenesis. Further understanding of the mechanisms involved in the activation of NF-kappa B in atherosclerosis could lead to important therapeutic applications especially as it relates to the impact of periodontitis.

Intracellular function in rehydrated lyophilized platelets.

This study aimed to evaluate the effect of cross-linking and lyophilization on intracellular signalling processes in rehydrated, lyophilized (RL) platelets, which are under development as a platelet substitute for transfusion. Exposure of RL platelets to thrombin resulted in enhanced phosphorylation of several proteins, including 18 kDa and 42 kDa kinase substrates that were shown to be the substrates of myosin light chain and protein kinase C respectively. Cross-linking and lyophilization depleted the platelets of free cytoplasmic ADP and ATP, but had less effect on protein-bound nucleotides. The surface membrane of RL platelets was found to be permeable to poly dT probes less than approximately 3 kDa in size; larger nucleotide probes and proteins did not penetrate the surface membrane. Taken together, our results indicate that RL platelets retain some of the haemostatic stimulus-response functions of fresh platelets and are capable of feedback amplification in coagulation.

Environmental tobacco smoke, cardiovascular disease, and the nonlinear dose-response hypothesis.

Two recent government reports have focused attention on the hypothesis that environmental tobacco smoke (ETS) exposure increases the risk of cardiovascular disease (CVD) in nonsmokers. The first report was published by the California Environmental Protection Agency (CAEPA) in 1997. The second report was issued in 1998 by the Scientific Committee on Tobacco and Health (SCOTH) in the United Kingdom. A meta-analysis of five large prospective epidemiology studies reports that the relative risk for actively smoking 20 cigarettes per day is 1.78. Active smoking exposes the smoker to approximately 16 times the ETS concentration, and 100- to 300-fold the total smoke dose experienced by a nonsmoker (Smith and Ogden, 1998, JAMA 280, 32-33.). Despite the much lower smoke exposure, these government reports estimate the relative risk for ischemic heart disease in ETS-exposed nonsmokers at 1.30 (CAEPA) and 1.23 (SCOTH). As an explanation for this nonlinear dose-response anomaly, platelet aggregation is proposed to be a plausible and quantitatively consistent mechanism. Herein, evidence is presented suggesting that this low-dose hypothesis is inconsistent with the biochemistry and physiology of platelets and with the literature on the cardiovascular pathology of active smoking. In addition, several important biases and confounders are ignored. These epidemiologic biases and confounders include the following: misclassification of smokers as nonsmokers; improper use of death certificates as surrogates for mortality statistics; underreporting of diabetes and hypertension in the relatives of smokers; and additional atherogenic risk factors in smoking households. Future field studies on ETS and CVD should emphasize proximal markers of risk for thrombosis in exposed nonsmokers. Proximal thrombogenic risk markers identified in field studies should be mechanistically examined under controlled exposure conditions.

Rap1b association with the platelet cytoskeleton occurs in the absence of glycoproteins IIb/IIIa.

Platelet membrane glycoproteins (GP) IIb/IIa and rap1b, a 21 kDa GTP binding protein, associate with the triton-insoluble, activation-dependent platelet cytoskeleton with similar rates and divalent cation requirement. To examine the possibility that GPIIb/IIIa was required for rap1b association with the cytoskeleton, experiments were performed to determine if the two proteins were linked under various conditions. Chromatography of lysates from resting platelets on Sephacryl S-300 showed that GPIIb/IIIa and rap1b were well separated and distinct proteins. Immunoprecipitation of GPIIb/IIIa from lysates of resting platelets did not produce rap1b or other low molecular weight GTP binding proteins and immunoprecipitation of rap1b from lysates of resting platelets did not produce GPIIb/IIIa. Finally, rap1b was associated with the activation-dependent cytoskeleton of platelets from a patient with Glanzmann's thrombasthenia who lacks surface expressed glycoproteins IIb and IIIa. Based on these findings, we conclude that no association between GPIIb/IIIa and raplb is found in resting platelets and that rap1b association with the activation-dependent cytoskeleton is at least partly independent of GPIIb/IIIa.

Incorporation of Rap 1b into the platelet cytoskeleton is dependent on thrombin activation and extracellular calcium.

Rap 1b is a 22-kDa low molecular mass GTP-binding protein which is both a member of the Ras superfamily and a substrate for cAMP-dependent protein kinase. Recently, evidence has been presented to show that Rap 1b is incorporated into the detergent-extracted cytoskeleton of platelets during thrombin-induced activation. The aims of this study were to compare the incorporation of Rap 1b into the detergent-extracted cytoskeleton after activation with different agonists, to examine the role of extracellular calcium on the incorporation of Rap 1b into the cytoskeleton, to investigate the relationship between the association of Rap 1b and other proteins with the cytoskeleton, and to determine the effect of phosphorylation of Rap 1b incorporation into the cytoskeleton. Platelets were activated with thrombin, A23187, phorbol myristate acetate, ADP, epinephrine, and collagen in the presence and absence of calcium. The time dependence of Rap 1b incorporation into the detergent-extracted cytoskeleton was then measured. When platelets were activated by thrombin in the presence of extracellular calcium, conditions which permit aggregation, incorporation of Rap 1b into the detergent-extracted cytoskeleton was biphasic. Approximately 20% of the total cellular Rap 1b incorporated into the cytoskeleton within seconds and was followed by a slower second phase of incorporation. In contrast, when platelets were activated by thrombin in the absence of calcium, conditions which inhibit aggregation, or by the other agents in the presence or absence of calcium, only the initial phase of Rap 1b incorporation into the cytoskeleton was measured. The incorporation of Rap 1b paralleled the incorporation of membrane glycoproteins (GP) IIb/IIIa and PECAM-1, but not the incorporation of pp60c-src. The GTPase-activating protein for Ras (Ras-GAP) did not associate with the detergent-extracted cytoskeleton. Two-dimensional isoelectric focusing SDS-polyacrylamide gel electrophoresis of the total cellular and cytoskeletal Rap 1b showed that unphosphorylated as well as phosphorylated isoforms of Rap 1b were incorporated into the cytoskeleton in the same molar ratio as was present in the intact cell. Furthermore, the rates of incorporation of phosphorylated and unphosphorylated Rap 1b into the cytoskeleton were similar. These experiments show that Rap 1b can regulate events that take place within seconds after activation, such as the initial formation of the cytoskeleton, as well as longer term changes in the cytoskeleton that occur in response to thrombin-induced aggregation. Furthermore, phosphorylation could modulate the (unknown) functions of Rap 1b as a component of the cytoskeleton.

Cytoskeletal interactions of Rap1b in platelets.

We have presented evidence that rap1b, a 22 kDa low molecular weight GTP binding protein, becomes associated with the cytoskeleton in thrombin-activated platelets. The initial incorporation is very rapid and occurs as fast as we can measure it. Thus, some rap1b is associated with the cytoskeleton as fast as it is formed. The remainder of the rap1b is incorporated more slowly. This biphasic incorporation of rap1b is similar to the incorporation of GPIIb/IIIa into the cytoskeleton, but no interaction between GPIIb/IIIa and rap1b could be demonstrated. Phosphorylation of rap1b by cAMP-dependent protein kinase did not inhibit its association with the cytoskeleton. We conclude that rap1b is one of an increasing number of proteins that associate with the cytoskeleton during cell activation. The function of rap1b in the cytoskeleton is unclear at this time. However, it is possible to speculate on potential roles. There is growing evidence that low molecular weight G proteins participate in the formation of multi-molecular aggregates. For example, p21rac promotes the assembly of a membrane-associated complex composed of NADPH oxidase, p47, and p67 and this complex is important for activation of NADPH oxidase in neutrophils. Similarly, in yeast, BUD1, a homolog of rap1, forms a complex with BUD5 (a homolog of GDI), BEMI, CDC24, and CDC42 (a homolog of G25K). This multi-protein aggregate may be important in cytoskeletal structure in yeast. In platelets, rad1b, which is membrane associated, may promote the assembly of a complex of proteins during cell activation and may localize this complex to the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

The localization of the cAMP-dependent protein kinase phosphorylation site in the platelet rat protein, rap 1B.

Rap 1B is a low molecular weight G protein which is phosphorylated by cAMP-dependent protein kinase. In order to identify the site of phosphorylation by cAMP-dependent protein kinase, purified rap 1B from human platelets was phosphorylated and subjected to limited proteolysis with trypsin. Single digestion fragment containing the phosphorylation site was obtained and purified by reversed-phase HPLC. Sequence analysis of the phosphorylated digestion fragment demonstrated that the sequence of the phosphorylation site was -Lys-Lys-Ser-Ser-. This sequence is near the carboxy terminus and is adjacent to the site of membrane attachment of the protein.

rap1B, a cAMP-dependent protein kinase substrate, associates with the platelet cytoskeleton.

rap1B is a member of the ras superfamily of low molecular weight GTP binding proteins which constitutes a focal point of GTP and cAMP signal transduction systems. Like other members of this superfamily, rap1B is membrane-associated in resting platelets, presumably through polyisoprenylation. The studies presented here were undertaken to determine the subcellular changes in rap1B localization during cell activation. Activated and unactivated platelets were fractionated by Triton X-100 lysis followed by differential centrifugation to obtain a 10,000 x g cytoskeleton fraction, a 100,000 x g membrane skeleton fraction, and a 100,000 x g supernatant fraction containing solubilized proteins. In unactivated platelets, rap1B was present in the 100,000 x g supernatant fraction. In contrast, in platelets activated with 1 unit/ml alpha-thrombin or with the calcium ionophore, A23187, rap1B was quantitatively recovered in the 10,000 x g cytoskeleton fraction. rap1B was absent from the 100,000 x g fraction containing the membrane skeleton and could not be detected in the 100,000 x g supernatant containing cytosolic proteins and solubilized membrane components. These results indicate that rap1B associates with the cytoskeleton during cell activation.

The development of a high-order Taylor expansion solution to the chemical rate equation for the simulation of complex biochemical systems.

A numerical method for evaluating chemical rate equations is presented. This method was developed by expressing the system of coupled, first-degree, ordinary differential chemical rate equations as a single tensor equation. The tensorial rate equation is invariant in form for all reversible and irreversible reaction schemes that can be expressed as first- and second-order reaction steps, and can accommodate any number of reactive components. The tensor rate equation was manipulated to obtain a simple formula (in terms of rate constants and initial concentrations) for the power coefficients of the Taylor expansion of the chemical rate equation. The Taylor expansion formula was used to develop a FORTRAN algorithm for analysing the time development of chemical systems. A computational experiment was performed with a Michaelis-Menten scheme in which step size and expansion order (to the 100th term) were varied; the inclusion of high-order terms of the Taylor expansion was shown to reduce truncation and round-off errors associated with Runge-Kutta methods and lead to increased computational efficiency.

Kinetic evidence that arachidonate-induced calcium efflux from platelet microsomes involves a carrier-type ionophoric mechanism.

Arachidonate, at concentrations up to 50 microM, induced dose-dependent calcium efflux from preloaded microsomes prepared from human platelets, but not from unilamellar egg phosphatidylcholine vesicles. Arachidonate-induced efflux from microsomes was not inhibited by indomethacin, 13-azaprostanoic acid, or catalase and superoxide dismutase, indicating that the release was due to arachidonate and not a metabolite. Linolenate (18:3, cis) and linoleate (18:2, cis) induced calcium efflux in a manner similar to arachidonate (20:4, cis), while arachidate (20:0), linolelaidate (18:2, trans), elaidate (18:1, trans), oleate (18:1, cis), stearate (18:0) and palmitate (16:0) had no effect. An experimental method was developed for distinguishing between carrier ionophore, small aqueous pore (i.e., calcium channel), or large aqueous pore (i.e., detergent effect) mechanisms in vesicular efflux systems in which calcium efflux occurs over a period of minutes. This development predicted that with a carrier ionophore mechanism, an increase in either internal or external calcium should competitively inhibit 45Ca efflux. In contrast, 45Ca efflux by diffusion through a small aqueous pore or a large aqueous pore should be measurably insensitive to variations in internal or external calcium. These predictions were experimentally verified in the platelet microsomal system using efflux agents with known mechanisms. Efflux of 45Ca by A23187, a calcium ion carrier ionophore, was sensitive to internal or external calcium competition, while alamethicin, a small aqueous pore channel model, and Triton X-100, a detergent which forms large aqueous pores, mediated 45Ca efflux which was measurably insensitive to variations in internal or external calcium concentration. Arachidonate-induced 45Ca efflux was inhibited by increasing either internal and external calcium concentration, suggesting that the fatty acid functions as a carrier ionophore. Arachidonate-induced 45Ca efflux was also inhibited with extravesicular Sr2+, but not Mn2+ or Ba2+. The dependence of the initial arachidonate efflux rate on arachidonate concentration showed that at least two arachidonates were contained in the calcium-carrier complex. These results are consistent with a model in which arachidonate (A) and an endogenous microsomal component (B) translocate calcium across the membrane through a carrier ionophore mechanism as part of a complex with a stoichiometry of A2B.Ca.

Thrombolamban, the 22-kDa platelet substrate of cyclic AMP-dependent protein kinase, is immunologically homologous with the Ras family of GTP-binding proteins.

Platelet inhibition by agents that increase intracellular levels of cAMP is associated with cAMP-dependent phosphorylation of specific intracellular proteins, including a membrane-associated 22-kDa microsomal protein called thrombolamban. In view of recent studies suggesting that platelets also contain 22-kDa GTP-binding proteins that are homologous with ras-encoded p21 proteins, the present work was undertaken to examine the possibility that thrombolamban and the Ras-like proteins were the same. Platelet microsomes were labeled with [gamma-32P]ATP and the labeled proteins were examined by autoradiography of sodium dodecyl sulfate/polyacrylamide gels. On Western blots of both one-dimensional and two-dimensional gels, thrombolamban immunoreacted with M90, a monoclonal antibody that recognizes the GTP-binding domain of Ras p21 proteins, but not with Y13-259, a monoclonal antibody that recognizes another domain and is specific for Ras proteins. Overlay experiments with unlabeled platelet microsomes demonstrated numerous low molecular weight proteins that bound [alpha-32P]GTP, although none could be identified as thrombolamban. Finally, thrombolamban was immunoprecipitated by M90. These studies show that thrombolamban is a low molecular weight protein that is immunologically related to the Ras family of GTP-binding proteins.

Cyclic AMP-dependent protein kinase does not increase calcium transport in platelet microsomes.

Cyclic AMP inhibits platelet activation, at least in part, by reducing intracellular levels of ionic calcium. Previous studies using platelet microsomal fractions have suggested that one mechanism for this effect is stimulation by cyclic AMP and its protein kinase of calcium uptake into microsomal storage sites. In the present study, the effect of cyclic AMP and its protein kinase on calcium uptake by microsomal membranes has been re-examined using the active catalytic subunit of cyclic AMP-dependent protein kinase. The catalytic subunit increased calcium uptake two-fold, but this effect was not inhibited by boiling the catalytic subunit or by recombination with the regulatory subunit of cyclic AMP-dependent protein kinase, conditions that inhibited catalytic subunit activity. Conversely, dialysis of the catalytic subunit preparation against low phosphate buffer, which did not inhibit catalytic subunit activity, inhibited the stimulation of calcium uptake by the catalytic subunit preparation. Finally, the addition of high phosphate buffer, similar in phosphate concentration to that of the catalytic subunit preparation, stimulated calcium uptake. We conclude that the catalytic subunit does not directly stimulate calcium uptake by platelet microsomes.

The identification of sarcoplasmic reticulum terminal cisternae proteins in platelets.

Two new proteins with apparent molecular masses of 53 kDa and 190 kDa have been identified in both sarcoplasmic reticulum and human blood platelets using a monoclonal antibody, FII1b5. The sarcoplasmic reticulum FII1b5 antigens were present in the terminal cisternae fraction, but were absent from light sarcoplasmic reticulum. The platelet and skeletal muscle proteins were not sensitive to digestion with endoglycosidase H under conditions that removed carbohydrate from the 53 kDa glycoprotein in sarcoplasmic reticulum or GPIIIa in platelet microsomes and did not bind 45Ca in a nitrocellulose overlay calcium-binding assay. These results distinguished the FII1b5 antigens from the 53 kDa glycoprotein and calsequestrin of sarcoplasmic reticulum. The 190 kDa platelet and sarcoplasmic reticulum proteins were extracted from membranes with high concentrations of NaCl, indicating that the high molecular mass FII1b5 antigens are peripherally associated with the bilayers. In contrast, the platelet and muscle 53 kDa proteins remained membrane-bound in the presence of high salt concentrations, suggesting that they are integral proteins.

cAMP-dependent protein kinase substrates in platelets. Evidence that thrombolamban, a 22,000 dalton substrate, and the Ca++-ATPase are not associated proteins.

Inhibition of platelet function by cAMP is due at least in part to a reduction in the agonist stimulated increase in cytoplasmic calcium during cell activation. This inhibition is also associated with cAMP-dependent phosphorylation of thrombolamban, a 22 kDa phosphoprotein which is present in the same membrane fraction as the calcium-dependent ATPase. Phosphorylation of this protein has been correlated with increased uptake of calcium by microsomal membranes. The present study was undertaken to examine the interaction of thrombolamban with the Ca++-ATPase in order to assess the possibility that the increased calcium uptake was by a direct effect of thrombolamban on Ca++-ATPase activity or that thrombolamban was a component of the Ca++-ATPase. Several approaches were utilized to assess the interaction of thrombolamban with the microsomal Ca++-ATPase. Gel filtration of labeled microsomes solubilized under non-denaturing conditions showed a major peak of radioactivity (Kav 0.64) corresponding to thrombolamban which was well separated from the Ca++-ATPase activity (Kav 0.09). Chemical cross-linking studies using partially purified thrombolamban and intact microsomes showed incorporation of the phosphoprotein into a 147,000 dalton complex. Indirect immunostaining with an anti-Ca++-ATPase antibody failed to demonstrate the Ca++-ATPase in the 147,000 dalton complex. Recombination of the phosphorylated thrombolamban with the Ca++-ATPase had no effect on Ca++-ATPase activity. These results indicate that, under the conditions used in these experiments, there was no apparent interaction between thrombolamban and the microsomal Ca++-ATPase. We conclude that thrombolamban is covalently bound to the Ca++-ATPase.

Partial purification and characterization of thrombolamban, a 22,000 dalton cAMP-dependent protein kinase substrate in platelets.

In preparations of human platelet microsomes, cyclic AMP-dependent protein kinase induced the rapid phosphorylation of a single protein that was electrophoretically identical to the 22,000 dalton protein (P22) phosphorylated by cAMP in intact platelets. Phosphorylation of the microsomal protein was maximal at one minute and was followed by slow dephosphorylation. Although the protein was associated with a microsomal fraction, it could be separated from the membrane by 2 M NaCl indicating that it was a peripheral protein. Molecular weight was estimated by NaDodSO4-PAGE and by gel filtration chromatography. The molecular weight estimated by NaDodSO4-PAGE was 22,400 daltons and was somewhat larger than the 16,000 molecular weight estimated by gel filtration in the presence of NaDodSO4. In the absence of NaDodSO4, the protein chromatographed as a 36,000 dalton form. The presence of the 36,000 dalton form was not dependent on the phosphorylation state of the protein. The partially purified protein contained phosphoserine, but no phosphothreonine or phosphotyrosine. Two dimensional NaDodSO4-PAGE and isoelectric focusing of the phosphorylated protein revealed isomers with pl values of 5.9 and 6.3. These studies indicate that the 22 kDa microsomal protein and P22 in intact platelets are the same protein and that the 22 kDa protein is tightly bound to the microsomal membrane although the nature of this binding and the microsomal component(s) to which it is bound remain to be determined. We conclude that the 22 kDa protein in platelet microsomes is structurally distinct from, but functionally similar to, phospholamban, the cAMP-dependent protein kinase substrate in muscle, and may play a similar role in calcium transport. Based on this similarity, it is proposed that the 22 kDa protein in platelets be called thrombolamban.

An investigation of functional similarities between the sarcoplasmic reticulum and platelet calcium-dependent adenosinetriphosphatases with the inhibitors quercetin and calmidazolium.

The platelet and skeletal sarcoplasmic reticulum calcium-dependent adenosinetriphosphatases (Ca2+-ATPases) were functionally compared with respect to substrate activation by steady-state kinetic methods using the inhibitors quercetin and calmidazolium. Quercetin inhibited platelet and sarcoplasmic reticulum Ca2+-ATPase activities in a dose-dependent manner with IC50 values of 25 and 10 microM, respectively. Calmidazolium also inhibited platelet and sarcoplasmic reticulum Ca2+-ATPase activities, with half-maximal inhibition measured at 5 and 4 microM, respectively. Both inhibitors also affected the calcium transport activity of intact platelet microsomes at concentrations similar to those which reduced Ca2+-ATPase activity. These inhibitors were then used to examine substrate ligation by the platelet and sarcoplasmic reticulum calcium pump proteins. For both Ca2+-ATPase proteins, quercetin has an affinity for the E-Ca2 (fully ligated with respect to calcium at the exterior high-affinity calcium binding sites, unligated with respect to ATP) conformational state of the protein that is approximately 10-fold greater than for other conformational states in the hydrolytic cycle. Quercetin can thus be considered a competitive inhibitor of the calcium pump proteins with respect to ATP. In contrast to the effect of quercetin, calmidazolium interacts with the platelet and sarcoplasmic reticulum Ca2+-ATPases in an uncompetitive manner. The dissociation constants for this inhibitor for the different conformational states of the calcium pump proteins were similar, indicating that calmidazolium has equal affinity for all of the reaction intermediates probed. These observations indicate that the substrate ligation processes are similar for the two pump proteins. This supports the concept that the hydrolytic cycles of the two proteins are comparable.