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+.     

Adenine nucleotide metabolism in human blood – important roles for leukocytes and erythrocytes

Adenosine diphosphate (ADP) released into blood induces platelet aggregation and contributes to hemostasis and thrombosis. Released ATP can also induce platelet aggregation and there is evidence that blood leukocytes and also erythrocytes play important roles in this. Rapid metabolism of ADP and ATP by endothelial cells is important in protecting platelets from their effects. Here we have performed a systematic investigation of adenine nucleotide metabolism in human blood and the involvement of blood cells. Conversion of ATP to ADP in blood was due almost exclusively to the presence of leukocytes; plasma, platelets and erythrocytes made little or no contribution. Mononuclear leukocytes (MNLs) and polymorphonuclear leukocytes (PMNLs) were equally effective. Conversion of ADP to AMP was also promoted by leukocytes, with no involvement of platelets or erythrocytes. Some ADP was also converted to ATP in blood, apparently via an enzyme present in plasma, but ATP was then rapidly removed by the leukocytes. Conversion of AMP to adenosine occurred via a plasma enzyme with little or no contribution from any cellular element. As expected, in blood the adenosine produced was removed very rapidly by erythrocytes and then converted to inosine and then hypoxanthine. In the absence of erythrocytes plasma supported only a slow conversion of adenosine to inosine and hypoxanthine, which was not influenced by platelets or leukocytes. This study has demonstrated that leukocytes and erythrocytes play a major role in adenine nucleotide metabolism in blood and that these cells, as well as endothelial cells, may be important determinants of the effects of ATP and ADP on platelets.     

The platelet defect in leukemia. Platelet ultrastructure, adenine nucleotide metabolism, and the release reaction.

The ultrastructure and adenine nucleotide metabolism of platelets from patients with acute leukemia were studied to elucidate possible mechanisms for the platelet dysfunction observed in this clinical setting. Nonstimulated (resting) platelets from leukemic patients varied greatly in size; exhibited marked variation in the number of alpha granules present per cell; had poorly delineated circumferential bands of microtubules; and often grossly dilated open channel systems or cytoplasmic vacuolization. The intracellular concentrations of ATP and ADP were significantly below normal, and the specific radioactivity of ATP and ADP of nonstimulated platelets in leukemia was equivalent to or exceeded that seen in stimulated normal platelets. Addition of ADP or collagen to platelets from leukemic patients was followed by retarded and incomplete shape change, delayed and incomplete centripetal migration of subcellular organelles, impaired degranulation, and the formation of loose aggregates composed of relatively few platelets. Stimulation of "leukemic" platelets with collagen led to the release of significantly subnormal amounts of ATP and ADP and no significant change in the specific radioactivity of the intracellular nucleotides. In contrast to the results in normal platelets, the conversion of ATP to inosine monophosphate and hypoxanthine in platelets in leukemia failed to increase significantly with collagen stimulation. The results indicate that abnormalities exist in the storage pool of adenine nucleotides and the release mechanism of platelets in acute leukemia. These defects appear to contribute to an impairment in the release reaction in these platelets. Many of the ultrastructural and metabolic defects seen in acute leukemia occur in platelets in preleukemia.     

亚低温对急性脑梗死大鼠脑组织Ca2+和Mg2+及兴奋性氨基酸与血浆内皮素变化的影响

背景急性脑梗死后脑组织内钙离子(Ca2+)、兴奋性氨基酸(excitatory amino acids,EAA)和血浆内皮素含量的升高及脑组织内镁离子(Mg2+)含量的降低都会加重脑组织的损害.给予亚低温干预后,观察急性脑梗死大鼠脑组织Ca2+和Mg2+及EAA及血浆内皮素的变化,了解亚低温的脑保护作用.目的探讨亚低温对大鼠急性脑梗死后脑组织内Ca2+及Mg2+和EAA、血浆内皮素变化的影响及意义.设计随机对照实验研究.单位武汉大学人民医院神经科.材料实验于2000-01/2000-12在武汉大学人民医院神经科实验室完成.将48只SD大鼠随机分为亚低温组及对照组,各组再分为4个亚组,每亚组6只.干预应用改良线拴法制备大鼠大脑中动脉梗死模型;亚低温组大鼠给予亚低温治疗,而对照组不作亚低温处理.分别于缺血后1,2,4,8 h处死.主要观察指标检测各个时段大鼠缺血区脑组织内Ca2+,Mg2+,EAA及血浆内皮素含量. 结果亚低温组大鼠缺血区脑组织内Ca2+,Mg2+,EAA及血浆内皮素含量随着缺血时间的延长仅轻度升高,Mg2+含量的下降也不明显,与对照组同时段比较有显著性差异(t=62.36～135.63,P＜0.01).结论亚低温能明显阻止实验动物脑缺血后缺血区脑组织内损伤性因子Ca2+,EAA和血浆内皮素含量的增高及保护性因子Mg2+的下降,具有脑保护作用.     

Reversibility of platelet P2Y12 inhibition by platelet supplementation: ex vivo and in vitro comparisons of prasugrel,clopidogrel and ticagrelor

Essentials

• Successful outcome of platelet transfusion depends on specific antiplatelet therapy in use.
• We assessed if ticagrelor, clopidogrel or prasugrel impacts on donor platelet activity ex vivo.
• Ticagrelor and/or its active metabolite in plasma or bound to platelets can inhibit donor platelets.
• This might compromise the effectiveness of platelet transfusion therapy.

Summary

Background

Platelet transfusion is the conventional approach to restore platelet function during acute bleeds or surgery, but successful outcome depends on the specific antiplatelet therapy. Notably ticagrelor is associated with inadequate recovery of platelet function after platelet transfusion. We examined whether plasma and/or platelets from ticagrelor‐treated patients influence donor platelet function, in comparison with clopidogrel and prasugrel.

Methods

Platelet transfusion was mimicked ex vivo by mixing naïve donor platelet‐rich plasma (PRP) or gel‐filtered platelets (GFP) in defined proportions with PRP, plasma or GFP from cardiovascular patients receiving standard care including medication with prasugrel, clopidogrel or ticagrelor (n = 20 each). Blood was taken 4 h after the previous dose. HLA2/HLA28 haplotyping let us distinguish net (all platelet) and individual patient/donor platelet reactivity in mixtures of patient/donor platelets, measured by flow cytometry analysis of ADP‐induced fibrinogen binding and CD62P expression.

Results

ADP responsiveness of donor platelets was dramatically reduced by even low (10%) concentrations of PRP or plasma from ticagrelor‐treated patients. Clopidogrel and prasugrel were associated with more modest donor platelet inhibition. GFP from ticagrelor‐treated patients but not patients receiving clopidogrel or prasugrel also suppressed donor GFP function upon mixing, suggesting the transfer of ticagrelor from patient platelets to donor platelets. This transfer did not lead to recovery of ADP responsiveness of patient's platelets.

Conclusion

Collectively, these observations support the concept that ticagrelor and/or its active metabolite in plasma or bound to platelets can inhibit donor platelets, which might compromise the effectiveness of platelet transfusion therapy.

     

Evidence that the P2x purinoceptor of the smooth muscle of the guinea pig vas deferens is an ATP4- receptor

In solutions containing Mg2+ and Ca2+, ATP is in equilibrium between the tetrabasic form (ATP4-) and its bidentate coordination complexes, i.e., MgATP2- and CaATP2-. We sought evidence to determine whether contractions of the smooth muscle of the guinea pig vas deferens to ATP are in response to ATP4- or its bidentate complexes. Contractions to ATP were elicited in seven modified Krebs-Henseleit solutions containing varied concentrations of free and total Mg2+ and Ca2+ to alter the concentration of ATP4- at given ATPtotal concentrations. As the concentration of Mg2+ increased the concentration of ATP required to stimulate contraction to an equivalent degree also increased. Regardless of the free or total Mg2+ and Ca2+ concentrations, response magnitude was generally correlated with [ATP4-]. This suggests that ATP4- is the agonist at the P2x purinoceptor of the guinea pig vas deferens. The potency of ATP4- is high; the threshold, occurring at approximately 1 nM ATP4-, is 1000-fold less than that for norepinephrine. The implications of ATP4- as agonist are discussed in relation to adenine nucleotide potency, metabolism and P2 purinoceptor classification.     

Decreased activity of the platelet Na+,K(+)-adenosine triphosphatase enzyme in allergic subjects

A pathogenic role and abnormal function have both been ascribed to the blood platelet in allergy, but the explanation for these observations is unknown. This study compared the cation-stimulated adenosine triphosphatase enzyme (ATPase) activities of platelets from allergic (n = 18), potentially allergic (asymptomatic, positive skin test, n = 5) and normal patients (n = 10), all of whom were without symptoms at the time of the study. Platelets were separated by centrifugation, were sonicated, and were assayed for cation-dependent ATPase activity by spectrophotometry. The mean Na+,K(+)-ATPase activity (in nanomoles per microgram protein per minute) of allergic subjects (0.94 +/- 1.28) was significantly lower than that of normal subjects (3.93 +/- 1.58). No Na+,K(+)-ATPase activity was detectable in platelets from eight of the allergic subjects. The Na+,K(+)-ATPase activity of potentially allergic subjects was intermediate between those of the allergic and normal subjects. A significant negative correlation (p less than 0.01) was observed between serum IgE levels and platelet Na+,K(+)-ATPase values, thus suggesting a relationship between the reduced platelet Na+,K(+)-ATPase and IgE immunoglobulin. No such differences were observed for the Ca+(+)- and Mg+(+)-stimulated ATPases. In vivo dysfunction of the plasma membrane Na+,K(+)-ATPase enzyme in allergic subjects could have profound effects on levels of intracellular cations and thus platelet activation and function.     

Obituaries

Platelet aggregation by adenosine diphosphate (ADP) is a self-limited and reversible process. An application of this phenomenon is described which allows removal of the platelets from large volumes of fresh platelet rich plasma (PRP) for the preparation of platelet concentrates. The macroscopic platelet clumps resulting from a concentration of 10 μgm ADP per ml of PRP are removed by centrifugation at 50 × g for 10 minutes. Resuspension of these platelets in 20 cc of native plasma results in a platelet concentrate that is 80 to 90 per cent as effective per unit as PRP in its ability to elevate the platelet count in recipients. Such concentrates are superior to concentrates prepared by other methods. The posttransfusion survival of ADP platelets compares favorably with the survival of platelets administered as PRP. There is evidence of minor sequestration but there is no apparent irreversible damage to platelets handled in this manner. Alkaline plasma and increase in plasma ionized calcium enhance the ADP aggregation and improve the efficiency of in vitro separation of platelets from PRP. However, the resulting concentrate is less effective in vivo, because of prolonged and slowly reversible clumping, and failure of these platelets to circulate.     

Evidence of a role for GTP in the potentiation of Ca(2+)-induced insulin secretion by glucose in intact rat islets.

Glucose initiates insulin secretion by closing K(+)-ATP channels, leading to Ca2+ influx (E1); it also potentiates Ca(2+)-induced secretion (E2) when the K(+)-ATP channel is kept open using diazoxide and depolarizing concentrations of K+ are provided. To examine the roles of purine nucleotides in E2, we compared the effects of glucose to those of the mitochondrial fuel monomethylsuccinate. Either agonist could induce E2 accompanied by significant increases in ATP, ATP/ADP ratio, and GTP/GDP ratio; GTP increased significantly only with glucose. Mycophenolic acid (MPA), an inhibitor of cytosolic GTP synthesis, markedly inhibited glucose-induced E2 (either in perifusions or in static incubations) and decreased GTP and the GTP/GDP ratio, but did not alter the ATP/ADP ratio. Provision of guanine (but not adenine) reversed these changes pari passu. In contrast, MPA had no effect on succinate-induced E2, despite generally similar changes in nucleotides. A similar lack of effect of MPA on E2 was seen with a second mitochondrial fuel, alpha-ketoisocaproic acid (KIC). However, in the absence of diazoxide and K+, MPA blunted the secretory effects of either glucose, succinate, or KIC. These studies suggest that GTP plays a role in both glucose and succinate or KIC-induced insulin secretion at a step dependent on mitochondrial metabolism and the K(+)-ATP channel. In addition to mitochondrial effects, glucose appears to have extramitochondrial effects important to its potentiation of Ca(2+)-induced insulin secretion that are also dependent on GTP.     

Hydrolysis of long-chain fatty acyl-CoA in homogenates of human blood platelets: the existence of a platelet palmitoyl-CoA hydrolase.

The existence of a very active long-chain fatty acyl-CoA hydrolase in homogenates of human blood platelets is reported. The highest activity was found with palmitoyl-CoA as the substrate. Palmitoyl-CoA hydrolase activity was not found in intact platelets indicating that the enzyme is localized within the platelet membrane. No palmitoyl-CoA hydrolase activity was found in fasting plasma. Mg2+, Mn2+, Ca2+ and Triton X-100 inhibited the palmitoyl-CoA hydrolase activity. Sulphydryl reagents had no effect, whereas high concentrations of D- and L-carnitine inhibited the activity. Carnitine palmitoyltransferase did not interfere with the assay of palmitoyl-CoA hydrolysis as the activity of carnitine-palmitoyl hydrolase was less than 1% of the palmitoyl-CoA hydrolase activity.     

Effect of common agonists on cytoplasmic ionized calcium concentration in platelets. Measurement with 2-methyl-6-methoxy 8-nitroquinoline (quin2) and aequorin

Because of controversy regarding the relationship of cytoplasmic ionized calcium concentration ([Cai2+]) to platelet activation, we studied the correlation of platelet aggregation and ATP secretion with [Cai2+] as determined by 2-methyl-6-methoxy 8-nitroquinoline (quin2) and aequorin in response to ADP, epinephrine, collagen, the Ca2+ ionophore A23187, and thrombin. Both indicators showed a concentration-dependent increase in [Cai2+] in response to all agonists except epinephrine when gel-filtered platelets were suspended in media containing 1 mM Ca2+. With epinephrine, a rise in [Cai2+] was indicated by aequorin, but not by quin2; [Cai2+] signals, aggregation, and secretion were suppressed by EGTA. ADP [0.5 microM] produced a rise in [Cai2+] that was registered by both aequorin and quin2 in platelets in Ca2+-containing media; addition of EGTA to the medium raised the threshold concentration of ADP to 5.0 microM for both indicators. Collagen produced progressive concentration-related increases in [Cai2+] and aggregation in aspirin-treated aequorin-loaded platelets. Quin2 failed to indicate a rise in [Cai2+]at lower collagen concentrations with EGTA or aspirin. [Cai2+] response to A23187 and thrombin was reduced by addition of EGTA to platelets loaded with either aequorin or quin2. With all five agonists in all conditions tested, aequorin [Cai2+] signals occurred at the same agonist concentration as that or lower than that which produced platelet shape change, aggregation, or secretion. Platelet activation was better correlated with changes in [Cai2+] indicated by aequorin than with the response of quin2, possibly because aequorin is more sensitive to local zones of [Cai2+] elevation.     

莫诺苷对二磷酸腺苷诱导兔血小板聚集钙离子的影响

目的观察莫诺苷对二磷酸腺苷(ADP)诱导兔血小板聚集后Ca²⁺+浓度的影响。方法利用钙离子荧光探针(Fura-2 AM)法,通过记录5 min 之内Fura-2 在激发波长340 nm和380 nm处的荧光强度比值,检测不同条件下ADP 诱导血小板聚集后Ca²⁺+的变化。结果与空白对照组相比,莫诺苷能显著抑制由ADP 诱导兔血小板聚集后Ca²⁺+的升高(P<0.001)。结论莫诺苷可能通过降低Ca²⁺+的上升达到抗ADP 诱导的兔血小板的聚集,从而改善体外血液流变学。     

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.     

Inihibition of human platelet aggregation by dipyridamole and two related compounds and its modification by acid glycoproteins of human plasma.

The inhibitory effect of dipyridamole (RA 8) and its two derivatives (RA 233 andSH 869) on platelet aggregation in platelet-rich plasma (PRP) AND IN SUSPENSIONSOF WASHED PLATELETS WAS EVALUATED USING 3 AGGRESSING STIMULI: ADP, thrombin, and collagen. Mean effective dose (ED'30) of RA 8 causing 50 percent inhibition of plateletaggregation of washed human platelets by ADP, collagen, or thrombin varied from 1.2 x 10'-7 to 1.8 x 10'-7M. On the other hand, RA 8 caused little inhibition aggregation in human PRP. RA 233 and SH 869 produced similiar degrees of inhibition ofplatelet aggregation in human PRP and in suspensions of washed human platelets.Platelet-poor plasma, fraction VI-acid glycoproteins, or purified alpha'1-acid glycoprotein complex was isolated by means of Sephadex G-25 gel filtration. It is postulated that the formation of this complex leads to the blocking of the capacity of RA 8to inhibit platelet aggragation. RA 233 and SH 869 had little capacity to form complexes with acid glycoproteins of human plasma. This may explain the effectiveness of these compounds in inhibiting platelet aggregation in PRP.     

Calcium transport and adenosine triphosphatase activities of erythrocyte membranes in congenital spherocytosis.

Calcium transport from red cells was measured in seventeen patients with congenital or hereditary spherocytosis (HS). The efflux remained at a lower level in resealed ghost cells of patients than in normal cells both in the presence and absence of adenosine triphosphate (ATP). We studied the activities of Ca2+,Mg2+-ATPase, ouabain-sensitive Na+,K+-ATPase, Mg2+-ATPase and Ca2+-(spectrin-)ATPase in cell membranes prepared by washing the cells with hypotonic medium. The mean +/-SD Ca2+,Mg2+-ATPase/Mg2+-ATPase of HS patients was 3.34 +/- 1.06, and 2.81 +/- 0.42 in control subjects. Na+,K+-ATPase/Mg2+-ATPase was 2.38 +/- 0.38 in HS cells compared to 2.01 +/- 0.41 in normal cells. Ca2+-ATPase/Mg2+-ATPase of HS membranes was 0.57 +/- 0.18 and the control value 0.43 +/- 0.08. These data indicate calcium retention in the erythrocytes of HS patients in spite of increases in Ca2+,Mg2+-ATPase activity in the majority of patients.     

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.     

Inhibitory effects of diltiazem on platelet activation caused by ionophore A23187 plus ADP or epinephrine in subthreshold concentrations

We examined the effects of the slow channel Ca++ blocker diltiazem on human platelet aggregation and TXA2 generation. Diltiazem inhibited platelet aggregation induced by 2 microM ADP or 5.5 microM epinephrine alone at 5 and 50 micrograms/ml (11.1 and 111 microM), respectively, and that induced by threshold concentrations of ADP or epinephrine at 0.2 to 1.0 micrograms/ml (0.4 to 2.2 microM). Platelet TXA2 generation stimulated by either ADP or epinephrine alone was inhibited by diltiazem in concentrations above the clinically achieved range (0.05 to 0.2 micrograms/ml, 0.1 to 0.4 microM). When PRP was stimulated with subthreshold concentrations of the Ca++ ionophore A23187 followed by subthreshold concentrations of ADP or epinephrine, a marked potentiation of platelet aggregation and TXA2 generation was observed. Incubation of PRP with diltiazem in a pharmacologic range resulted in marked reduction in ionophore A23187-induced potentiation of platelet activity caused by ADP or epinephrine. In other experiments, diltiazem was found to have no effects on PGI2-induced platelet aggregation inhibition. On the basis of these data, we conclude that (1) Ca++ flux across the platelet membrane stimulates platelet activity of subthreshold concentrations of ADP or epinephrine and (2) diltiazem in therapeutic concentrations reduces platelet activation induced by ionophore A23187 plus ADP or epinephrine, most likely by inhibiting Ca++ flux. These effects of diltiazem may not be observed in the therapeutic range if aggregatory concentrations of ADP or epinephrine alone are used.     

Effect of zinc and other cations on the release of the eosinophil cationic protein

The eosinophil cationic protein, ECP, is a unique eosinophil granule constituent, which is released extracellularly after exposure of the eosinophils to a non-phagocytosable surface such as complement-coated Sephadex beads. The ECP is released to some extent even in the absence of Ca2+ and Mg2+, though both these cations augment the release reaction tested alone, and an optimal release is observed only in the presence of 2 mmol/l Ca2+ and 2 mmol/l Mg2+ in the medium. Zn2+ at concentrations from 0.25-4.0 mmol/l inhibited the release of ECP in a dose-dependent fashion, with or without Ca2+ and Mg2+ in the medium. Mn2+ had dual effects, stimulating the ECP release in the absence of Mg2+ and Ca2+, and inhibiting the release in the presence of these cations. Li1+ caused minor inhibition of ECP release, but only in the absence of Ca2+ and Mg2+. The inhibitory effect of Zn2+ was immediate and reversible after washing of the cells, suggesting that the inhibition is due to interaction with the plasma membrane functions.     

Initiating and potentiating role of platelets in tissue factor-induced thrombin generation in the presence of plasma: subject-dependent variation in thrombogram characteristics

Summary.  The hemostatic activity of plasma is determined by platelet activation and coagulation, which processes are mutually stimulatory. We studied this interaction by measuring the cleavage of fluorescent thrombin substrate in platelet-rich plasma (PRP), using the calibrated thrombogram method. In freshly isolated human plasma, thrombin formation triggered by tissue factor was fully dependent on the presence of platelets. It was abolished by annexin A5, indicating dependence on phosphatidylserine (PS) exposure at activated platelets. Comparison of plasmas from various subjects showed considerable interindividual variation in total amount of thrombin generation, regardless of whether platelets or PS-containing phospholipids were present. Integrin α_IIbβ₃ antagonists and ADP receptor blockage, but not aspirin, decreased the rate of thrombin generation (thrombin peak level) and extended the time of onset. Platelet inhibition with cAMP-elevating agents decreased the thrombin-forming rate, but surprisingly shortened the onset time. Stimulation of platelets with agonists of Gi/q-coupled receptors and, to a larger extent, with collagen or Ca²⁺-ionophore increased the rate of thrombin generation and shortened its onset. In PRP from donors with low and high generation, platelet inhibitors and activators were similarly effective. Taken together, these results indicate that, in tissue factor-triggered PRP, PS exposure on activated platelets regulates both onset and rate of thrombin generation. However, coagulant activity rather than platelet activation determines the total amount of thrombin formed, i.e. the endogenous thrombin potential. Thus, kinetics of thrombin generation in PRP are controlled by platelet inhibitors and agonists, but the process is restricted in amount by the subject-dependent variation in coagulation.     

Liquid-chromatographic measurements of inosine, hypoxanthine, and xanthine in studies of fructose-induced degradation of adenine nucleotides in humans and rats

We applied a sensitive, precise liquid-chromatographic method of analysis for inosine, hypoxanthine, and xanthine to the study of fructose metabolism in humans and in rats. In the rat, intravenous loading with fructose induced, within minutes, substantial increases in the concentrations of inosine, hypoxanthine, and xanthine in plasma and urine. In plasma, these concentrations peaked after 5 min, then practically disappeared within 10 min. As expected, the fructose-induced increase in hypoxanthine was greatly amplified by pretreating the rats with allopurinol, an inhibitor of xanthine oxidase. In a healthy human subject, intravenous administration of fructose also induced prompt, substantial, and rapidly reversing increases in the concentrations of these metabolites of adenine nucleotides in plasma. The finding that fructose induced almost-immediate increases in the plasma concentrations of inosine, hypoxanthine, and xanthine is consistent with previous studies in rats, in which parenteral administration of fructose induced almost-immediate decreases of total adenine nucleotides (ATP + ADP + AMP) in the liver, and increased concentrations of uric acid and allantoin in the plasma.