Effect of epinephrine on fibrinogen receptor exposure by aspirin-treated platelets and platelets from concentrates in response to ADP and thrombin

Synergistic effects between agonists on platelet aggregation have long been appreciated. Recently epinephrine was reported to induce maximal aggregation of aspirin-treated platelets when combined with ADP or thrombin, and to increase fibrinogen binding of non-aspirin treated platelets stimulated with low doses of ADP. The present study extends these observations to correlate fibrinogen binding in response to various combinations of ADP, epinephrine, and thrombin with platelet aggregation and 14C-serotonin release using aspirin-treated platelets as well as platelets from stored concentrates. When fresh platelets were stimulated with epinephrine (5 microM) together with either ADP (10 microM) or thrombin (150 mU/ml), fibrinogen binding increased by 180% compared to binding observed in response to ADP or thrombin alone. This was accompanied by enhanced platelet aggregation, but no increase in 14C-serotonin release. While both ADP and epinephrine potentiated the aggregation and fibrinogen binding of stored platelets in response to high doses of thrombin (150 mU/ml), maximal aggregation was achieved only with thrombin (150 mU/ml) and epinephrine (5 microM) in combination. The data thus suggest that 1) epinephrine induces maximal aggregation of aspirin-treated platelets stimulated with thrombin or ADP by significantly enhancing fibrinogen receptor exposure independently of the cyclooxygenase-mediated release reaction; 2) epinephrine stimulates platelets by a mechanism different from that of thrombin or ADP; and 3) as demonstrated by others, the ability of platelets from stored concentrates to aggregate and to bind fibrinogen in response to ADP can be enhanced by epinephrine, and, in addition, these platelets can aggregate and bind fibrinogen maximally when stimulated with combinations of epinephrine and thrombin.     

凝血酶的神经保护作用

凝血酶在脑血管病中的作用日益受到重视。已有研究证实,凝血酶具有双向作用,小剂量可产生神 经保护作用,大剂量则具有细胞毒性作用。小剂量凝血酶预处理可减轻大剂量凝血酶对神经细胞的毒性作 用,其作用机制与MAPK信号转导途径、HSP表达等有关。     

Thrombin binding to platelets from hypercholesterolaemic rats

Platelets from rats made hypercholesterolaemic with a diet enriched with milk fat and cholesterol and containing taurocholate to promote hypercholesterolaemia aggregated more extensively to a low concentration of thrombin than platelets from rats given a milk fat-enriched diet containing sitosterol. Total and specific binding of thrombin to platelets from hypercholesterolaemic rats was significantly greater than in controls when expressed per mg platelet protein, per mumol platelet cholesterol, or per unit relative surface area. Total and specific binding of thrombin per platelet were not different between the groups. However, platelets from hypercholesterolaemic rats had less protein and cholesterol, were smaller and had less surface area than control platelets; platelet cholesterol content expressed per mg platelet protein was not different. Thus, the increase in thrombin-binding to the smaller platelets from hypercholesterolaemic rats during the first 10 s after its addition may be responsible, at least in part, for the hypersensitivity of these platelets to thrombin.     

Platelet and Thrombin Activity Following Cardiac Catheterization Despite Treatment with Aspirin

Thromboembolic complications are reported to occur in up to 0.5–2% of left cardiac catheterizations and angiographies. Activation of the hemostatic system may contribute to their onset. To prevent platelet and thrombin activity during catheterization, aspirin or systemic heparin are often used in addition to heparinized flush solutions. We investigated whether aspirin alone can prevent platelet and thrombin activity induced by catheterization in ten consecutive patients (nine males, mean 50 ± 8 years) undergoing elective left cardiac catheterization after at least 5 days of oral aspirin (75–300 mg/d). Anticoagulant drugs were not given. Peripheral venous samples were drawn before, immediately after (time 0), and at 15, 60, and 180 minutes after the procedure for measurement of thrombin–antithrombin (TAT), prothrombin fragment 1.2 (F 1.2), fibrinopeptide A (FPA), and -thromboglobulin (-TG). TAT, F1.2, and FPA increased significantly at time 0 compared with both before and 180 minutes after the procedure (P < 0.04); -TG values were higher at time 0 compared with 180 minutes later (P = 0.01). TAT levels were related to those of FPA (r = 0.66; P = 0.0003), F1.2 (r = 0.35; P = 0.01), and -TG (r = 0.37; p = 0.04). Thus, routine left cardiac catheterization is associated with transient, systematically detectable, activation of coagulation and platelets, despite aspirin therapy. Newer antiplatelet agents may be more effective in preventing hemostatic activation induced by catheterization.     

Immunogold staining of microtubules in resting and activated platelets

A circumferential microtubule is known to support the discoid form of resting platelets, but its fate following exposure of the cells to aggregating agents is uncertain. The present study has employed an immunocytochemical approach to follow the fate of the circumferential microtubule in activated platelets. Monoclonal antibodies to tubulin and to vinculin and a polyclonal antibody to actin were incubated with isolated microtubule coils and stained with staphylococcal protein A coupled to immunogold in order to test their specificity. Thin sections of glycolmethacrylate embedded platelets before and after exposure to thrombin for 15, 30 and 60 s were stained with antibodies to tubulin and actin. Immunogold particles showed a high specificity for isolated MT coils stained for tubulin, modest intensity for actin, and none for vinculin. Gold particles were randomly distributed in thin sections of resting and activated platelets stained for actin. Immunogold was limited to the circumferential microtubule in resting platelets and constricted coils in thrombin-activated cells. The number of gold particles in areas of cytoplasm away from microtubules in platelets stained with antitubulin antibody increased slightly following thrombin activation, but the change was not significant. Results support the concept that microtubule coils supporting the discoid form of resting platelets do not dissolve following exposure of the cells to potent agonists.     

Porcine von Willebrand factor and thrombin induce the activation of c-Jun amino-terminal kinase (JNK/SAPK) whereas only thrombin induces activation of extracellular signal-related kinase 2 (ERK2) in human platelets

The interaction of platelets with subendothelial von Willebrand factor (VWF), especially under high shear stress, is considered to be the first activation step which primes platelets for subsequent haemostatic events. The signalling cascade which results from the interaction of VWF and its receptor GPIbIX has only been partially defined. Mitogen-activated protein kinases (MAPKs) are a family of downstream transmembrane signalling serine-threonine kinases and have been demonstrated to be present and functional in platelets; these include the extracellular signal-related kinases (ERKs), c-Jun amino-terminal kinases (JNKs) and p38 MAPK. Previously, we showed that p38 MAPK was not required in VWF-induced human platelet activation. It is not known whether VWF-dependent platelet activation involves the activation of the JNK and ERK family of signalling molecules. This report demonstrates that porcine von Willebrand factor (pVWF) induced a sustained and stable JNK activation measurable by 1 min after activation. Thrombin also induced JNK activation assessed at 1 min after activation. In contrast to thrombin, pVWF did not induce ERK2 activation at any time point tested. To ensure that ERK activation was unnecessary for pVWF-dependent platelet activation, we functionally inhibited ERK-dependent signalling with PD98059, a potent and selective inhibitor of the MAP kinase kinase (MEK-1), which is the upstream kinase of ERK1 and ERK2. Although PD98059 inhibited ERK2 activation in platelets, it had no effect on pVWF- or thrombin-induced platelet alpha or lysozomal granule release, modulation of membrane glycoprotein CD41, microparticle formation, platelet shape change or platelet agglutination. It is concluded that pVWF and thrombin induced JNK activation, but whereas thrombin induced ERK2 activation VWF did not; functional ERK2 activity was also not required for pVWF- or thrombin-dependent platelet activation.     

Plasmin accelerates platelet-dependent prothrombinase formation without activating the platelets

Patients with acute myocardial infarction who undergo thrombolytic therapy may shortly thereafter present evidence for increased platelet activation and thrombin activity, and recurrent thrombosis. This study investigated whether plasmin activates platelets and prothrombin in recalcified platelet-rich plasma (RPRP) to cause (at least in part) these side-effects of thrombolytic therapy. Plasmin (0.1 and 1.0 CU/ml) addition to RPRP with 1 μ M r-tick anticoagulant peptide (the latter a factor Xa inhibitor which abrogates prothrombin activation by prothrombinase at the concentration used) resulted in no change in the concentration of prothrombin fragment 1 + 2, or in the expression of GMP-140, the resting and activated GP IIb–IIIa conformers, and GPIb on platelets. Thus, plasmin neither activates platelets nor prothrombin in RPRP. However, plasmin accelerated platelet activation and secretion, and prothrombin fragment 1 + 2 production in RPRP. When combined with 1 μ M r-tick anticoagulant peptide and 1 or 10 n M α-thrombin to RPRP, plasmin also increased the number of GMP-140 molecules expressed/platelet without enhancing α-thrombin binding to the platelets. Additionally, plasmin accelerated prothrombin activation when it was added to washed platelets resuspended in factor V depleted plasma simultaneously with 10 m M CaCl₂, 10 n M α-thrombin for 10 s (to activate platelets and platelet factor V), followed by 4 μ M hirudin and 1 n M factor Xa. Thus, plasmin potentiates the platelet release reaction in response to α-thrombin (probably by increasing the availability of factor V on the platelets) to enhance prothrombin activation in RPRP. These actions of plasmin may contribute to the increased platelet activation and thrombotic side-effects that can occur after thrombolytic therapy.     

Thrombin receptor expression and function in large granular lymphocyte proliferative disorders

Summary. It has recently been shown that peripheral blood NK-cells and a fraction of T-cells which co-express CD16 and either CD56 or CD57 express the platelet type thrombin receptor. Large granular lymphocytes exhibit a T- or NK-cell phenotype, and therefore these results raise the possibility that thrombin and its receptor may be involved in the biology of large granular lymphocytes in health and disease. It is difficult, however, to perform functional studies using normal blood as a source of large granular lymphocytes because the small fraction of large granular lymphocytes cannot be separated from other lymphocytes in numbers sufficient for most in vitro experiments. Therefore patients with large granular lymphocyte proliferative disorders have been screened in order to identify a population of cells enriched in large granular lymphocytes that express the thrombin receptor. Expression of the receptor was analysed in polyclonal and clonal large granular lymphocyte proliferative disorders. Using flow cytometry, it was found that the proportion of thrombin receptor positive large granular lymphocytes varied from 3% to 86%. Northern analysis indicated a high level of expression of mRNA in a clonal expansion of large granular lymphocytes that stained positively for the receptor by flow cytometry. Thrombin was found to act as a chemotactic stimulus for large granular lymphocytes from a polyclonal expansion with high numbers of thrombin receptor positive cells. At an optimal concentration of 10^-9 m the chemotactic response to thrombin was roughly equivalent to that obtained with the potent chemoattractant 1-oleoyl 2-acetyl glycerol. These findings suggest that thrombin may play a role in the recruitment of large granular lymphocytes in sites of inflammation.     

Thrombin binding to platelets defines functional receptors: inhibition of thrombin-induced platelet activation by catalytically-inactivated thrombin

It has been widely questioned as to whether the observed binding of a-thrombin to intact platelets defines receptors coupled to signal transduction or merely thrombin binding sites. We have now shown that at α-thrombin concentrations sufficient to induce a full shape change response without aggregation (0.1 nM), PPACK-thrombin (that is, α-thrombin treated with the irreversible active site inhibitor D-phenylalanyl-L-prolyl-L-arginine chloromethylketone) dose-dependently inhibits platelet shape change (IC(50)～70 nM), the concomitant increases in [Ca(2+)Ii (IC(50)～75 nM) and ATP secretion (IC(50)～50 nM). Since PPACK-thrombin competes fully in the binding of a-thrombin to high, moderate and low affinity sites on intact platelets, these results show that this binding defines functional receptors coupled to platelet activation.     

Complement (C3) binding to platelets in autoimmune thrombocytopenia

Summary. In view of conflicting reports on the occurrence of complement binding to platelets in idiopathic autoimmune thrombocytopenic purpura (AITP) we performed measurements of platelet bound C3 in patients with AITP who had elevated levels of platelet bound IgG. Using a quantitative antiglobulin consumption technique 38 out of 42 patients were found to have fixed abnormally large amounts of C3 to their platelets, and a significant positive correlation between the amounts of platelet bound IgG and C3 was shown to exist. In additional experiments antibody eluates were prepared from AITP platelets and were shown to cause the fixation of C3 to normal donor platelets in vitro. Taken together these findings strongly suggest that the C3 binding in AITP is specifically related to the disease process.     

Microparticles adhere to collagen type I, fibrinogen, von Willebrand factor and surface immobilised platelets at physiological shear rates

Microparticles (MPs), shed during the storage of platelets, support blood coagulation and could be helpful in restoring the haemostatic system in thrombocytopenic patients. The mechanisms by which MPs support haemostasis under flow conditions were investigated. Fluorescent-labelled MPs were perfused at shear rates of 100 and 1000/s over surfaces coated with collagen, fibrinogen, von Willebrand factor (VWF) or surface-adherent platelets. Adhesion was monitored in real-time by fluorescence microscopy. In addition, thrombin-antithrombin (TAT) complex formation was measured in flowing thrombocytopenic blood. MPs attained the capacity to firmly adhere to collagen, VWF, fibrinogen and surface-adherent platelets at high and low shear rate. Antibodies against glycoprotein Ibalpha and alpha(IIb)beta(3) were used to demonstrate the specificities of these interactions. The addition of MPs to thrombocytopenic blood did not affect platelet adhesion. TAT complex formation was increased in the presence of MPs in capillaries coated with fibrinogen, but not on collagen fibres. We confirmed that MPs adhere to a damaged vascular bed in vivo after infusion in denuded arteries in a mouse model. MPs have platelet-like adhering properties and accelerate thrombin generation. These properties strongly support the notion that MPs can be beneficial in maintaining normal haemostasis when platelet function is impaired or reduced, as in thrombocytopenic patients.     

Myristoylated alanine-rich C kinase substrate phosphorylation is involved in thrombin-induced serotonin release from platelets

Stimulation of platelets by thrombin induces protein kinase C (PKC) activation, phosphorylation of pleckstrin, aggregation and serotonin release. Here, we demonstrate that, in human platelets, thrombin stimulation also induced phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) and serotonin release in intact and digitonin-permeabilized platelets. MARCKS is known to bind actin and cross-link actin filaments, and this is inhibited by PKC-evoked MARCKS phosphorylation. MARCKS phosphorylation and serotonin release in response to increasing concentrations of thrombin have a similar EC50 and time course and, in permeabilized platelets, peptide MPSD, with an amino acid sequence corresponding to the phosphorylation site domain of MARCKS, blocked both responses. However, pleckstrin and myosin light chain phosphorylations were not modified. Ala-MPSD, in which the four serine residues of MPSD were substituted by alanines was ineffective. The results suggest a role for MARCKS in platelet secretion. The fact that pleckstrin phosphorylation has a different time course and was not modified in the presence of MPSD when MARCKS phosphorylation and serotonin release were inhibited would suggest either that pleckstrin phosphorylation is unrelated to secretion or that it might only be involved upstream in the events leading to secretion.     

Assays for phenotypic and functional characterization of cryopreserved platelets

Platelet concentrates for transfusion have a limited shelf-life, and cryopreservation offers a means of extending platelet shelf-life for at least 2 years. Cryopreservation, however, has some disadvantages, as platelets can be damaged during the freezing and thawing process. Consequently the phenotype of cryopreserved platelets is very different to that of freshly collected, liquid stored platelets. To obtain a reliable overview of cryopreserved platelet quality and function, specific testing methodologies should be considered.

Whilst light transmission aggregometry (LTA) is the gold standard for measuring in vitro platelet reactivity in liquid stored platelets, it is not suited for cryopreserved platelets, as they retain little response to typical platelet agonists, even at high doses. Instead, phenotypic characterization by flow cytometry in combination with global assays of coagulation provides a clearer delineation of the phenotype and function of cryopreserved platelets. With increasing international uptake of platelet cryopreservation, it is important to recognize the need for appropriate measures of platelet quality and function.     

Dynamic nature of thrombin generation,fibrin formation,and platelet activation in unstable angina and non-Q-wave myocardial infarction

Background Thrombin and platelets are directly involved in arterial thrombosis, typically occurring at sites of atherosclerotic plaque rupture among patients with acute coronary syndromes. Understanding the dynamic nature of pathologic thrombosis has important clinical implications.Methods Fibrinopeptide A (FPA), thrombin-antithrombin complexes (TAT), and prothrombin activation fragment 1.2 (F1.2), plasma markers of fibrin formation (thrombin activity) and thrombin generation, and platelet activation, determined by the recognition of a surface-expressed platelet -granule protein, P-selectin, using flow cytometry, were measured in 36 consecutive patients with unstable angina and non-Q-wave myocardial infarction participating in the Thrombolysis In Myocardial Ischemia (TIMI) III B trial.Results Thrombin generation (TAT 12.1 ± 17.8 ng/ml vs. 3.4 ± 1.0 ng/ml; F1.2 0.19 ± 0.14 nmol/1 vs. 0.12 ± 0.8 nmol/1), fibrin formation (FPA 15.8 ± 23.5 ng/ml vs. 7.5 ± 2.3 ng/ ml), and platelet activation) 10.6 ± 2.4% vs. 2.5 ± 2.0%) were increased significantly in patients compared with healthy, age-matched controls (p < 0.01). Fibrin formation, represented by plasma FPA levels, did not correlate with the percentage of activated platelets (r=– .10, p=0.69). Thrombin generation and platelet activation also did not correlate. A statistically insignificant trend between TAT and platelet activation was observed (r=.42, p=0.07); however, even with TAT levels in excess of 20 ng/ml (nearly sixfold greater than normal healthy controls) platelet activation was increased by only 1.7-fold.Conclusions Thrombin generation, fibrin formation, and platelet activation are increased modestly among patients with unstable angina and non-Q-wave myocardial infarction. Despite the involvement of platelets and coagulation proteins in arterial thrombotic processes, their relative contributions may vary, providing a pathophysiologic basis for the dynamic expression of disease and response to treatment observed commonly in clinical practice.     

Newborn platelets: Lower levels of protease-activated receptors cause hypoaggregability to thrombin

Newborn platelets show in vitro hypoaggregability to thrombin. Sensitivity of platelets to such a potent agonist is crucial for a functional clot formation. Nevertheless, newborns have an excellent hemostasis. We wanted to investigate the reason for this impairment by comparatively analysing levels of receptors known to be involved in thrombin signaling in newborn and adult platelets. Platelets of adult and cord blood were isolated, washed, and lysed. Resulting protein samples were separated by SDS-PAGE and blotted on nitrocellulose membranes. Receptors were visualized using immunodetection and evaluated densitometrically. Thrombin receptor activating peptide induced platelet aggregation was measured in citrated whole blood on a Multiplate analyzer. Statistical analysis was performed using SPSS 16.0. Significantly lower levels of protease-activated receptors (PAR1, PAR4) and higher levels of glycoprotein Ibα (GPIbα) were found in newborn platelets as compared to adult platelets. Platelet aggregation was lower in newborn samples than in adult controls and values correlated with the corresponding PAR levels. Our results suggest that lower levels of protease-activated receptors contribute to the poor thrombin induced aggregation observed with newborn platelets, which can not be compensated by higher levels of GPIbα.     

Thrombin activatable fibrinolysis inhibitor activation and bleeding in haemophilia A

Individuals with haemophilia A exhibit bleeding tendencies that are not always predicted by their factor (F)VIII level. It has been suggested that bleeding in haemophilia is due not only to defective prothrombin activation but also aberrant fibrinolysis. Thrombin activatable fibrinolysis inhibitor (TAFI) activation was measured in tissue factor (TF)-initiated blood coagulation in blood samples of 28 haemophiliacs and five controls. Reactions were quenched over time with FPRck and citrate and assayed for TAFIa and thrombin-antithrombin (TAT). The TAFIa potential (TP), TAFI activation rate and the TAFIa level at 20 min (TAFIa(20 min)) was extracted from the TAFI activation progress curve. In general, the time course of TAFI activation follows thrombin generation regardless of FVIII activity and as expected the rate of TAFI activation and TP decreases as FVIII decreases. The magnitude of TP was similar among the control subjects and subjects with <11% FVIII. In severe subjects with <1% FVIII at the time of blood collection, the TAFIa(20 min) was inversely and significantly correlated with haemarthrosis (-0.77, P = 0.03) and total bleeds (-0.75, P = 0.03). In all cases, TAFIa(20 min) was more strongly correlated with bleeding than TAT levels at 20 min. Overall, this study shows that TAFI activation in whole blood can be quantified and related to the clinical bleeding phenotype. Measuring TAFIa along with thrombin generation can potentially be useful to evaluate the differential bleeding phenotype in haemophilia A.     

脑出血后凝血酶的作用与抗凝血酶治疗

文章介绍了凝血酶的结构、其在中枢神经系统产生和代谢特征、在脑出血后脑水肿形成中的重要作用及其可能机制,内源性和外源性凝血酶抑制剂,脑出血抗凝血酶治疗用药的初步临床试验结果。随着凝血酶在脑出血后作用研究的深入,抗凝血酶治疗可能是防治出血性脑损伤最有希望的措施之一。     

Should platelets be radiolabelled in plasma medium?

Because of the low labelling efficiency of platelets with In-111 oxine in plasma containing media, most investigators have labelled platelets in plasma-free media. However, labelling of platelets with In-111 oxine in plasma-free media may result in either: irreversible damage leading to rapid clearance from the circulation and permanent hepatic sequestration; or reversible damage with transient hepatic sequestration without affecting the in vivo survival. Since human platelets can be labelled with In-111 oxine in plasma in sufficient efficiency for routine clinical use, they should be labelled in plasma whenever possible. The use of newer In-111 chelates, tropolone and mercaptopyridine-N-oxide, which label platelets with high efficiency in plasma as well as in plasma-free media, may offer another alternative.     

Presence of the seven transmembrane thrombin receptor on human tumour cells: effect of activation on tumour adhesion to platelets and tumour tyrosine phosphorylation

Thrombin-treated tumour cells enhance their adhesion to platelets, fibronectin and von Willebrand factor in vitro , and enhance their pulmonary metastasis in mice in vivo . A unique seven transmembrane spanning thrombin receptor has recently been cloned which is activated following thrombin proteolysis of the N-terminal end of the receptor with exposure of a tethered ligand. An N-terminal 14-mer (SFLLRNPNDKYEPF) or 6-mer (SFLLRN) of the tethered ligand can serve as a thrombin receptor activation peptide (TRAP) by mimicking the action of thrombin on platelets, endothelial cells and smooth muscle cells. We have examined six human tumour cell lines for their response to TRAP, for the presence of this thrombin receptor mRNA by RT-PCR, protein by immunoblot and for their in vitro and in vivo response to TRAP. All six cell lines contain the receptor mRNA, and when treated with 100 μ m 6-mer TRAP or 1 u/ml thrombin increase their adhesion to platelets 2–3-fold. Four of the six cell lines undergo tyrosine phosphorylation within 30 s to 1 min after exposure to 6-mer TRAP or thrombin. Thus tumour cells respond to thrombin via activation of their seven transmembrane spanning thrombin receptor.