The binding of fibrinogen to platelets in diabetes mellitus

Platelets from diabetics are known to be more sensitive in vitro to a variety of aggregating agents, to produce more prostaglandin endoperoxides and thromboxane and to bind more 125I-fibrinogen than platelets from normal controls Fibrinogen binding to platelets is a pre-requisite for platelet aggregation. Previous studies suggested a role for prostaglandins and/or thromboxane A2 in the exposure of fibrinogen receptors on platelets. The present study compares fibrinogen receptors on platelets. The present study compares fibrinogen binding to hyperaggregable platelets from diabetic patients and to normal platelets when prostaglandin/thromboxane formation is suppressed by aspirin. It was found that pre-treatment with aspirin reduced collagen or thrombin-induced binding to platelets from non-retinopathic diabetics to the values seen in controls. By contrast, aspirin did not normalize binding to platelets obtained from retinopathic diabetics. The combination of aspirin with apyrase (an ADP scavenger) almost completely inhibited binding and aggregation of platelets from normal controls or non-retinopathic diabetics exposed to collagen or thrombin, whereas it only partially affected binding and aggregation of platelets from retinopathics. By using a monoclonal antibody (B59.2) to the platelet receptor for fibrinogen, we determined that this receptor was quantitatively and qualitatively the same on platelets from normal controls and diabetics. We conclude that increased fibrinogen binding and hyperaggregability of platelets from non-retinopathic diabetics is related to their capacity to form more prostaglandin endoperoxides/thromboxane than normal platelets. In contrast, hyperaggregability and increased binding of platelets from retinopathics appear at least partly related to a mechanism independent of ADP release and thromboxane synthesis.     

Mechanisms underlying ATP-induced Ca2+ mobilization in human neutrophils

The effect of ATP on Ca2+ mobilization in human neutrophils was examined by using fura-2 as a Ca2+ indicator. ATP (0.1-100 microM) caused a significant [Ca2+]i increase in a concentration-dependent manner. The [Ca2+]i signal comprised an initial rise followed by a plateau. Removal of external Ca2+ diminished the peak value of the [Ca2+]i signal. In Ca2+-free medium, pretreatment with an endoplasmic reticulum Ca2+ pump inhibitor, thapsigargin, prevented ATP from releasing Ca2+. In contrast, thapsigargin still increased [Ca2+], after pretreatment with 10 microM ATP. These results indicate that 10 microM ATP released Ca2+ mainly from thapsigargin-sensitive stores. Adding 3 mM Ca2+ induced a concentration-dependent increase in [Ca2+]i after pretreatment with ATP or thapsigargin in Ca2+-free medium, suggesting ATP induced Ca2+ influx via capacitative Ca2+ entry. ATP (10 microM)-induced Ca2+ release was abolished by inhibiting phospholipase C with 2 microM U73122, indicating that inositol-1,4,5-trisphosphate (IP3) mediates ATP-induced Ca2+ release. Conversely, ATP-induced [Ca2+]i increase was abolished by activating protein kinase C (PKC) with 10 nM phorbol myristate acetate (PMA), but was not altered by inhibiting PKC with 2 microM GF 109203X. This implies ATP-induced [Ca2+]i increase is a PMA-linked event. Together, the results suggest ATP increases [Ca2+]i in human neutrophils by releasing Ca2+ from IP3-coupled, thapsigargin-sensitive Ca2+ stores, and inducing Ca2+ influx via the process of capacitative Ca2+ entry. The ATP-induced Ca2+ signal is a PMA-linked event.     

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+.     

Hypersensitivity to ADP of platelets from diabetic rats associated with enhanced fibrinogen binding

Platelets from diabetic humans and animals are hypersensitive to ADP. The hypersensitivity to ADP of platelets from diabetic rats occurs independently of activation of the arachidonate pathway or release of dense granule contents. During platelet aggregation by ADP, fibrinogen binds to its receptor on platelets. We examined if the hypersensitivity to ADP of platelets from diabetic rats is associated with enhanced early binding of fibrinogen to its receptor on these platelets. Fibrinogen association with platelets from rats with spontaneous or streptozotocin-induced diabetes was significantly greater 10 s or 1 min after addition of ADP (10 microM) than with platelets from their corresponding control rats. Since enhanced fibrinogen association occurred with platelets from insulin-treated rats with spontaneous diabetes, and from rats with streptozotocin-induced diabetes that did not receive insulin, the enhanced fibrinogen binding is likely due to the diabetic state rather than to the administration of insulin or the mechanism responsible for the diabetes. Therefore, enhanced early fibrinogen association with platelets from diabetic rats is associated with their hypersensitivity to ADP.     

Aggregation of chymotrypsin-treated thrombasthenic platelets is mediated by fibrinogen binding to glycoproteins IIb and IIIa

Previous experiments demonstrated that chymotrypsin, but not adenosine diphosphate (ADP), exposed fibrinogen binding sites on platelets from patients with Glanzmann's thrombasthenia. Three of these patients have been reexamined, and previous observations were confirmed. The quantity of iodine 125-labeled glycoprotein IIb (GPIIb) and glycoprotein IIIa (GPIIIa) on the platelets of these patients was considerably less than normal but was detectable by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography. The amount of residual GPIIb and GPIIIa as measured by binding studies with radiolabeled monoclonal antibodies was between 3% and 12% of the normal value. Platelet suspensions from these patients did not aggregate with fibrinogen and did not bind 125I-fibrinogen on stimulation with ADP. However, incubation of these platelets with chymotrypsin or pronase resulted in fibrinogen binding and platelet aggregation. Monoclonal antibodies specific for the GPIIb-GPIIIa complex blocked both the fibrinogen binding and the aggregation of enzyme-treated platelets. The treatment of washed platelets of a fourth thrombasthenic patient with ADP or with chymotrypsin failed to result in fibrinogen binding and aggregation. However, the level of GPIIb and GPIIIa on these platelets as measured by a Western blot technique and by monoclonal antibody binding amounted to less than 0.35% to 0.5% of normal values. In conclusion, fibrinogen binding sites exposed on thrombasthenic platelets by chymotrypsin are derived from GPIIb-GPIIIa molecules. Aggregation of chymotrypsin-treated thrombasthenic platelets by fibrinogen appears to represent a sensitive test for detection of functionally active GPIIb-GPIIIa complex on the platelet surface.     

化学诱导剂对血小板钙离子浓度变化的影响

目的探讨化学诱导剂作用下不同人群血小板钙离子浓度的变化。方法（1）以不同浓度的ADP、凝血酶、胶原、花生四烯酸、5-HT激活血小板，用流式细胞仪观察20例正常健康人血小板钙离子浓度变化。（2）用ADP、胶原、花生四烯酸、5-HT激活血小板，用流式细胞仪观察心肌梗死患者及高血压患者（各20例）血小板钙离子浓度的变化。（3）用花生四烯酸激活血小板，用流式细胞仪观察20例心肌梗死患者口服阿司匹林前后血小板钙离子浓度的变化。结果（1）ADP、瑞斯托酶素、胶原、花生四烯酸、5-HT血小板激活剂均可导致血小板钙离子浓度升高（P〈0．05），而且在一定范围内，钙离子浓度的升高幅度随激活剂的浓度升高而增加。没有观察到凝血酶导致血小板钙离子浓度升高。（2）同样激活剂刺激下，心肌梗死患者和高血压患者血小板钙离子浓度升高的幅度均高于正常健康人（P〈0．05）。（3）服用阿司匹林后心肌梗死患者血小板钙离子浓度下降的幅度低于正常健康人（P〈0．05）。结论化学诱导剂对血小板的激活过程与钙离子浓度的变化密切相关；心肌梗死、高血压患者可能存在血小板钙通道活性增高；钙离子能反应服用阿司匹林后血小板的功能状态，检测血小板钙离子对阿司匹林的疗效监测有一定的价值．     

Intracellular Ca2+ behavior and activation of calpain-I in activated platelets]

A relationship between intracellular Ca2+ concentration ([Ca2+]i) and calpain-I activation and change in subcellular localization of the enzyme in activated platelets were investigated. The [Ca2+]i exhibited a biphasic response after stimulation with thrombin. Activation of calpain-I was measured by determination of the appearance of active 76 and 78 kDa forms accompanying the disappearance of the 80 kDa form, the inactive form, on immunoblots. Calpain-I was activated dependent on the extent of the initial elevation of [Ca2+]i. For maximum activation (60%) 300-500 nM [Ca2+]i was required and half-maximal activation occurred at 160-220 nM [Ca2+]i. The active 76 kDa form was observed only in the fraction containing subcellular organelles and plasma membrane of activated platelets. It was demonstrated that the localization of calpain-I was changed from the cytosol to the membrane and calpain-I was activated on the membrane by Ca2+, elevated through the initial elevation after activation of platelets.     

Mechanism of bradykinin-induced Ca2+ mobilization in murine proximal tubule epithelial cells

Despite the recognized physiological role of bradykinin (BK) in the kidney in maintaining glomerular and tubule function and its role in pathological states such as endotoxemia, diabetes, and other diseases, relatively little is known about the mechanisms by which BK can impact kidney function. Furthermore, the signaling of BK receptors in the murine nephron has not been fully characterized. The present studies were undertaken to examine BK-stimulated Ca(2+) signaling using Fura-2 in the murine proximal tubule epithelial cell line TKPTS. BK produced a concentration-dependent rise in intracellular Ca(2+) ([Ca(2+)])(i) (pEC(50) = 8.39 +/- 0.04). Selective antagonists showed the rise in [Ca(2+)](i) was mediated through B2 receptors. The rise in [Ca(2+)](i) was rapid and reversible and was maximally stimulated at 1 microM (697 +/- 70 nM above basal level of 115 +/- 6 nM). Studies with thapsigargin and EGTA showed Ca(2+) mobilization was dependent on two events: release and influx. Both U73122 (1-[6-[[17-beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione) [a phospholipase C (PLC) inhibitor] and genistein (a tyrosine kinase inhibitor) partially inhibited BK-stimulated rise in [Ca(2+)](i). When combined, both agents produced a further decrease, suggesting multiple pathways for PLC activation may be involved. The ability of Ni(2+) to inhibit influx indicated the activation of a Ca(2+) release-activated channel (CRAC). Ca(2+) mobilization did not seem to be affected by cyclic nucleotides or protein kinase C. In summary, the TKPTS murine proximal tubule cell line expresses functional B2 receptors linked to Ca(2+) mobilization that is dependent on phospholipase C and activation of CRAC.     

Specific correction of impaired acid hydrolase secretion in storage pool-deficient platelets by adenosine diphosphate.

Storage pool-deficient (SPD) platelets, which have decreased amounts of dense-granule and/or alpha-granule constituents, contain normal amounts of lysosomal acid hydrolases, but in some cases exhibit impaired secretion of these enzymes. We examined this impaired secretion response in SPD patients with varying extents of granule deficiencies, and determined the effects of added dense-granule constituents. Acid hydrolase secretion was impaired in patients with severe dense-granule deficiencies, but not in patients with lesser dense-granule deficiencies, including those with alpha-granule deficiencies as well. When dense-granule constituents (ADP, ATP, serotonin, Ca+2, pyrophosphate) were added to gel-filtered platelets, ADP, but none of the other constituents, completely corrected the impairment of thrombin and A23187-induced secretion in SPD platelets. The concentration of ADP required to normalize thrombin-induced secretion varied markedly, from 0.01 to 10 microM, among the individual patients. Fixation of platelets with formaldehyde before centrifugation did not prevent the enhancement of secretion by ADP. Excess ATP, which acts as a specific antagonist of ADP-mediated responses, completely blocked this enhancement of secretion in SPD platelets by ADP, and partially inhibited acid hydrolase secretion induced by low, but not high, concentrations of thrombin in normal platelets as well. Treatment of normal platelets with acetylsalicylic acid in vivo, but not in vitro, produced an impairment of acid hydrolase secretion similar in extent to that in SPD platelets, but which could not be completely corrected by added ADP. One possible explanation of these results is that the impairment of acid hydrolase secretion may be secondary to the dense-granule deficiency in SPD platelets, and that secreted ADP may potentiate the lysosomal secretion response in normal platelets as well.     

Mechanism of myricetin stimulation of vascular L-type Ca2+ current

An in-depth analysis of the mechanism of the L-type Ca(2+) current [I(Ca(L))] stimulation induced by myricetin was performed in rat tail artery myocytes using the whole-cell patch-clamp method. Myricetin increased I(Ca(L)) in a frequency-, concentration-, and voltage-dependent manner. At holding potentials (V(h)) of -50 and -90 mV, the pEC(50) values were 4.9 +/- 0.1 and 4.2 +/- 0.1, respectively; the latter corresponded to the drug-apparent dissociation constant for resting channels, K(R), of 67.6 microM. Myricetin shifted the maximum of the current-voltage relationship by 10 mV in the hyperpolarizing direction but did not modify the threshold for I(Ca(L)) or the T-type Ca(2+) current. The Ca(2+) channel blockers nifedipine, verapamil, and diltiazem antagonized I(Ca(L)) in the presence of myricetin. Myricetin increased the time to peak of I(Ca(L)) in a voltage- and concentration-dependent manner. Washout reverted myricetin effect on both current kinetics and amplitude at V(h) of -90 mV while reverting only current kinetics at V(h) of -50 mV. At the latter V(h), myricetin shifted the voltage dependence of inactivation and activation curves to more negative potentials by 6.4 and 13.0 mV, respectively, in the mid-potential of the curves. At V(h) of -90 mV, myricetin shifted, in a concentration-dependent manner, the voltage dependence of the inactivation curve to more negative potentials with an apparent dissociation constant for inactivated channels (K(I)) of 13.8 muM. Myricetin induced a frequency- and V(h)-dependent block of I(Ca(L)). In conclusion, myricetin behaves as an L-type Ca(2+) channel agonist that stabilizes the channel in its inactivated state.     

The regulatory mechanism of free Ca2+ concentration in activated platelets]

The change in intracellular Ca2+ concentration ([Ca2+]i) following platelet stimulation results from mobilization, influx and restoration of Ca2+. To determine whether inositol 1,4,5 trisphosphate (IP3) is involved in Ca2+ influx, the relationship between IP3 formation (IP3) and Ca2+ influx ( delta [Ca2+]i) was investigated in platelets stimulated wtih various agonists (thrombin, ADP, PAF, STA2, etc). The ratio of IP3 to delta [Ca2+]i varied among the agonists, although delta [Ca2+]i was increased, depending on the amount of agonist. Furthermore, in spite of the similar delta [Ca2+]i, IP3 was smaller at 20 degrees C compared with that at 37 degrees C in thrombin-stimulated platelets. These results indicate that Ca2+ influx in platelets might be regulated by receptor-operated Ca2+ channel rather than by an IP3 mediated mechanism. As for Ca2+ restoration, calpain was demonstrated to play a role through Ca(2+)-ATPase activation by limited proteolysis.     

三七皂甙单体2A-1-1对人血小板聚集和钙内流的作用

目的观察三七皂甙单体2A-1-1对人血小板聚集和钙内流的影响,并探讨其对受体操纵性钙通道的作用.方法比浊法测定血小板聚集;Fura-2/Am荧光探针双波长测定细胞胞浆游离钙浓度,观察2A-1-1、硝苯地平、SK＆F96365对二磷酸腺苷(ADP)、环匹阿尼酸(CPA)介导的人血小板钙内流的变化.结果硝苯地平(20μmol/L)不能抑制ADP诱导的血小板聚集,不能抑制ADP或CPA介导的血小板钙内流;SK＆F96365(20μmol/L)可以抑制ADP诱导的血小板聚集,抑制率为59.83%;SK＆F96365(15μmol/L)可以抑制CPA和ADP介导的钙内流;2A-1-1(5,10,20,μmol/L)可抑制ADP诱导的血小板聚集,抑制率分别为47.06%,53.47%,71.52%;2A-1-1(10,20 μmol/L)可抑制CPA和ADP介导的钙内流.结论三七皂甙单体2A-1-1能抑制人血小板聚集,抑制血小板受体操纵性钙通道,从而抑制钙内流,有抗血小板作用.     

Interaction of prostaglandins with adenosine diphosphate induced increase in cytosolic free calcium in human platelets.

The interaction of prostaglandins with changes in cytosolic Ca2+ concentration ([Ca2+]) and aggregation of human platelets induced by adenosine diphosphate (ADP) were investigated. Cytosolic [Ca2+] was measured with the fluorescent dye Quin2. Addition of ADP (0.25-2.5 mumol l-1) to platelet suspensions produced a dose dependent increase in cytosolic [Ca2+] from a basal level of 51 +/- 1 nmol l-1 to maximum levels exceeding 1 mumol l-1 and induced platelet aggregation. Chelation of extracellular calcium with 100 mumol l-1 EGTA markedly reduced the increase in cytosolic [Ca2+] induced by 0.25 mumol l-1 ADP, while pretreatment with the calcium entry blocker verapamil was without effect. Stimulation of cyclic AMP with prostaglandins (PGD2, PGE1, PGE2, PGI2, but not PGF2 alpha) and forskolin, or incubation with dibutyryl-cAMP, inhibited the rise in cytosolic [Ca2+] and platelet aggregation following ADP. We conclude that prostaglandins inhibit the increase in cytosolic [Ca2+] and aggregation of human platelets induced by ADP, probably by stimulation of cyclic AMP generation, thereby opposing the mechanism by which ADP increases cytosolic [Ca2+] and subsequently induces platelet aggregation.     

3-O-acetyl-11-keto-boswellic acid decreases basal intracellular Ca2+ levels and inhibits agonist-induced Ca2+ mobilization and mitogen-activated protein kinase activation in human monocytic cells

Previously, we showed that 11-keto-boswellic acid and 3-O-acetyl-11-keto-BA (AKBA) stimulate Ca(2+) mobilization and activate mitogen-activated protein kinases (MAPKs) in human polymorphonuclear leukocytes (PMNLs). Here, we addressed the effects of boswellic acids on the intracellular Ca(2+) concentration ([Ca(2+)](i)) and on the activation of p38(MAPK) and extracellular signal-regulated kinase (ERK) in the human monocytic cell line Mono Mac (MM) 6. In contrast to PMNLs, AKBA concentration dependently (1-30 microM) decreased the basal [Ca(2+)](i) in resting MM6 cells but also in cells where [Ca(2+)](i) had been elevated by stimulation with platelet-activating factor (PAF). AKBA also strongly suppressed the subsequent elevation of [Ca(2+)](i) induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP), PAF, or by the direct phospholipase C activator 2,4, 6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide, but AKBA failed to prevent Ca(2+) signals induced by thapsigargin or ionomycin. Suppression of Ca(2+) homeostasis by AKBA was also observed in primary monocytes, isolated from human blood. Moreover, AKBA inhibited the activation of p38(MAPK) and ERKs in fMLP-stimulated MM6 cells. Although the effects of AKBA could be mimicked by the putative phospholipase C (PLC) inhibitor U-73122 (1-[6-[[17beta-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione), AKBA appears to operate independent of PLC activity since the release of intracellular inositol-1,4,5-trisphosphate evoked by 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide was hardly diminished by AKBA. Inhibitor studies indicate that AKBA may decrease [Ca(2+)](i) by blocking store-operated Ca(2+) and/or nonselective cation channels. Together, AKBA interferes with pivotal signaling events in monocytic cells that are usually required for monocyte activation by proinflammatory stimuli. Interruption of these events may represent a possible mechanism underlying the reported anti-inflammatory properties of AKBA.     

Highly constrained cyclic (S,S) -CXaaC- peptides as inhibitors of fibrinogen binding to platelets

The Arg-Gly-Asp RGD motif of adhesive proteins is recognized by the activated platelet integrin alpha(IIb)beta3. Binding of fibrinogen (Fg) to activated alpha(IIb)beta3 causes platelet aggregation and thrombus formation. Highly constraint cyclic (S,S) -CXaaC- containing peptides incorporating the (S,S) -CDC- and (S,S) -CRC- motifs were tested for their ability to inhibit platelet aggregation and Fg binding. Our results suggest that the above cyclic scaffolds stabilize a favorable structure for the antiaggregatory activity (IC50-values ranged from 1.7 to 570 microm). The peptides inhibited Fg binding with IC50-values up to 30-fold lower than those determined for the inhibition of the adenosine diphosphate (ADP)-induced platelet aggregation. Importantly, peptides (S,S) PSRCDCR-NH2 (peptide 11) and (S,S) PRCDCK-NH2 (peptide 10) did not inhibit PAC-1 binding to the activated platelets at a concentration in which they completely inhibited Fg binding. Moreover, (S,S) PSRCDCR-NH(2) (peptide 11), one of the more active peptides, inhibited ADP-induced P-selectin exposure. By contrast, peptide (S,S) Ac-RWDCRC-NH2, incorporating the inverse (S,S) -DCRC- sequence (peptide 16), failed to inhibit P-selectin exposure whereas at the same concentration, it effectively inhibited PAC-1 and Fg binding. It is concluded that peptides containing the (S,S) -CDC- as well the (S,S) -CRC- sequences, exhibit a broad range of activities toward platelets, and could be helpful tools for elucidating the structural interaction of Fg with the integrin receptor alpha(IIb)beta3, in its activated form. Furthermore, the (S,S) -RCDC- sequence can be used as a scaffold for developing potent non-RGD-like Fg-binding inhibitors.     

Ca2+ mobilization in saphenous vein smooth muscle cells derived from patients with primary varicosity

BACKGROUND: Human primary varicosity is associated with 'weakness' of the vein wall. We investigated whether the reduced responsiveness of varicose veins to physiological vasoconstrictors might result from impaired Ca2+ mobilization in venous smooth muscle. MATERIALS AND METHODS: The hypothesis was tested in cells derived from phenotypically different vein segments that were obtained from the inguinal saphenous vein (tissue with incompetent valves), the distal portion of the long saphenous vein just above the medial ankle (clinically healthy tissue), and from a tributary to the long saphenous vein just below the knee (incompetent and overtly varicose tissue). Saphenous vein from patients undergoing cardiac surgery served as control. Cytosolic free Ca2+ levels ([Ca2+]i) were determined with the fura-2 method in cultured medial smooth muscle cells of third to sixth passage (21-23 measurements per tissue derived from five controls and seven patients). RESULTS: Angiotensin II (10 nmol L-1 to 10 mumol L-1) induced a significantly (P < 0.05) smaller rise in [Ca(2+)1i response in cells derived from incompetent or varicose segments (approximatley 70 nmol L-1) than in cells derived from clinically healthy vein (approximately 130 nmol L-1) or controls (approximately 170 nmol L-1). Likewise, the effect of endothelin-1 (100 nmol L-1) on [Ca2+]i was considerably less in cells derived from segments with incompetent valves or from varicose vessel segments than in cells derived from control patients (P < 0.05). In organ baths, endothelium-denuded strips of varicose vessels contracted significantly less in response to these agonists than clinically healthy segments from the same patient. CONCLUSIONS: The reduced contractility of diseased human varicose veins in response to angiotensin II and endothelin-1 involves impaired Ca2+ mobilization.     

The mobilization of arachidonic acid in platelets exposed to thrombin or ionophore A23187. Effects of adenosine triphosphate deprivation.

In studies conducted with human gel-filtered platelets, we have found: (a) that the release of serotonin and transfer of [3H]arachidonic acid from phosphatidylcholine and phosphatidylinositol to plasmalogen phosphatidylethanolamine which are associated with the activation of platelets by thrombin are both strongly dependent upon the presence of metabolic ATP; (b) that serotonin release and arachidonic acid mobilization in labeled phosphatides are promoted by the calcium ionophore A-23187 in media free of calcium ions; (c) that inhibitors of ATP synthesis, while leading to impairment of the release reaction induced by ionophore, do not inhibit ionophore-stimulated mobilization of arachidonic acid. We conclude that the activation of phospholipase A2 responsible for freeing arachidonic acid from platelet phosphatides is solely dependent upon the increased cytoplasmic levels of calcium ions promoted by either ionophore or, in an energy-dependent fashion by thrombin. Phospholipase activation is not a function of latent hydrolytic activity made available by the release reaction.     

Increased leakage of sarcoplasmic reticulum Ca2+ contributes to abnormal myocyte Ca2+ handling and shortening in sepsis

OBJECTIVE: Changes in cardiac function due to sepsis have been widely reported. However, the underlying mechanisms remain poorly understood. In the mammalian heart, myocyte function and intracellular calcium homeostasis are closely coupled. In this study we tested the hypothesis that alterations in cardiac calcium homeostasis due to sepsis underlie the observed myocyte dysfunction. DESIGN: Randomized prospective animal study. SETTING: Research laboratory. SUBJECTS: Male Sprague-Dawley rats weighing 250-275 g. INTERVENTIONS: We induced sepsis by cecal ligation and puncture in the rat, which mimics the type of infection caused by perforation of the intestine in humans. MEASUREMENTS AND RESULTS: Forty-eight hours after cecal ligation and puncture, isolated cardiac ventricular cardiomyocytes demonstrated a 57% decreased peak systolic [Ca]. The time constant of the Ca transient increased 71% and 57% in myocytes obtained 24 hrs and 48 hrs after cecal ligation and puncture, respectively. The average shortening of cardiomyocytes 48 hrs after cecal ligation and puncture was significantly decreased. To investigate the cellular mechanisms of altered Ca transients and myocyte shortening, we measured Ca sparks, the spontaneous local Ca release events in cardiomyocytes at resting states. The Ca spark frequency progressively increased in myocytes 24 hrs and 48 hrs after cecal ligation and puncture. The total activity of sparks also increased compared with sham-operated animals. The overall leakage of sarcoplasmic reticulum Ca in resting states was increased in sepsis and resulted in reduced sarcoplasmic reticulum Ca content. CONCLUSIONS: Abnormal Ca leakage from the sarcoplasmic reticulum contributes significantly to the depressed myocyte shortening in sepsis. In the future, modalities that prevent this Ca leakage may prove beneficial in the treatment of sepsis-induced myocyte shortening.