Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The antiphospholipid (aPL) syndrome is an autoimmune condition that is marked by recurrent pregnancy losses and/or systemic vascular thrombosis in patients who have antibodies against phospholipid/co-factor complexes. The mechanism(s) for pregnancy losses and thrombosis in this condition is (are) not known. Annexin A5 is a potent anticoagulant protein, expressed by placental trophoblasts and endothelial cells, that crystallizes over anionic phospholipids, shielding them from availability for coagulation reactions. We previously presented data supporting the hypothesis that aPL antibody-mediated disruption of the anticoagulant annexin A5 shield could be a thrombogenic mechanism in the aPL syndrome. However, this has remained a subject of controversy. We therefore used atomic force microscopy, a method previously used to study the crystallization of annexin A5, to image the effects of monoclonal human aPL antibodies on the crystal structure of the protein over phospholipid bilayers. In the presence of the aPL monoclonal antibodies (mAbs) and beta(2)-GPI, the major aPL co-factor, structures presumed to be aPL mAb-antigen complexes were associated with varying degrees of disruption to the annexin A5 crystallization pattern over the bilayer. In addition, measurements of prothrombinase activity on the phospholipid bilayers showed that the aPL mAbs reduced the anti-coagulant effect of annexin A5 and promoted thrombin generation. These data provide morphological evidence that support the hypothesis that aPL antibodies can disrupt annexin A5 binding to phospholipid membranes and permit increased generation of thrombin. The aPL antibody-mediated disruption of the annexin A5 anticoagulant shield may be an important prothrombotic mechanism in the aPL syndrome.
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PMID:Human monoclonal antiphospholipid antibodies disrupt the annexin A5 anticoagulant crystal shield on phospholipid bilayers: evidence from atomic force microscopy and functional assay. 1293 61

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 alphaIIbbeta3 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.
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PMID:Initiating and potentiating role of platelets in tissue factor-induced thrombin generation in the presence of plasma: subject-dependent variation in thrombogram characteristics. 1500 66

The platelet procoagulant response requires a sustained elevation of the intracellular Ca2+ concentration, [Ca2+]i, causing exposure of phosphatidylserine (PS) at the outer surface of the plasma membrane. An increased [Ca2+]i also activates Ca2+-dependent K+ channels. Here, we investigated the contribution of the efflux of K+ ions on the platelet procoagulant response in collagen-thrombin-activated platelets using selective K+ channel blockers. The Gardos channel blockers clotrimazol, charybdotoxin, and quinine caused a similar decrease in prothrombinase activity as well as in the number of PS-exposing platelets detected by fluorescence-conjugated annexin A5. Apamin and iberiotoxin, inhibitors of other K+ channels, were without effect. Only clotrimazol showed a significant inhibition of the collagen-plus-thrombin-induced intracellular calcium response. Clotrimazol and charybdotoxin did not inhibit aggregation and release under the conditions used. Inhibition by Gardos channel blockers was reversed by valinomycin, a selective K+ ionophore. The impaired procoagulant response of platelets from a patient with Scott syndrome was partially restored by pretreatment with valinomycin, suggesting a possible defect of the Gardos channel in this syndrome. Collectively, these results provide evidence for the involvement of efflux of K+ ions through Ca2+-activated K+ channels in the procoagulant response of platelets, opening potential strategies for therapeutic interventions.
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PMID:Reversible inhibition of the platelet procoagulant response through manipulation of the Gardos channel. 1674 Dec 54

The role of phospholipid platelet membrane and tissue factor in thrombin generation and thrombus formation is accepted. In the present study we have explored antithrombotic action of strategies aimed to block exposure of negatively charged phospholipids and we compared effects with those obtained through tissue factor or a direct thrombin inhibition. Type III collagen was exposed to flowing blood (5 min, 300 s(-1)). Effects of inhibition of platelet deposition by annexin A5 (ANXA5), hirudin (HIR) or by an antibody against tissue factor (TF) were evaluated. Prothrombin fragment F1 + 2 (F1 + 2) was monitored. Pre-incubation of whole blood with HIR or ANXA5 resulted in a statistically significant reduction of platelet deposition (12.2 +/- 0.6% in control experiments vs. 8.3 +/- 0.4% and 8.5 +/- 0.5%, respectively, P < 0.05). A similar decrease was found when blood was incubated with an antibody against TF. Furthermore, ANXA5 and HIR inhibited the recruitment of platelets into forming aggregates. The height of platelet aggregates generated was decreased in the presence of HIR or ANXA5, but only incubation with both inhibitors reached levels of statistical significance. The presence of ANXA5 or HIR decreased levels of F1 + 2 suggesting a reduced activation of the coagulation system. In our experimental studies, the inhibitory potential of ANXA5 on platelet-thrombus formation was as effective as that of a direct thrombin inhibitor, as HIR, or an antibody against TF. Negatively charged phospholipids exposed on activated platelets potentiate the formation of platelet aggregates on a collagen surface and further suggest that inhibition of platelet procoagulant activity might be a specific target for antithrombotic drugs.
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PMID:Antithrombotic action of annexin V proved as efficient as direct inhibition of tissue factor or thrombin. 1691 46

In the Bernard-Soulier syndrome (BSS), the giant platelets are said to have increased phosphatidylserine (PS) surface exposure in the resting state and shortened survival in the circulation. When normal platelets are activated, they undergo many biochemical and morphological changes, some of which are apoptotic. Herein, we investigated apoptotic-like events in BSS platelets upon activation, specifically, PS exposure, microparticle (MP) formation, cell shrinkage, and loss of mitochondrial inner membrane potential (DeltaPsi(m)). Platelets from two unrelated BSS patients were examined in whole blood; agonists used were collagen, thrombin, PAR1- or PAR4-activating peptides (APs), or combinations of collagen with thrombin, and the PAR-APs. Flow cytometry was used to measure PS exposure (annexin A5 binding), platelet-derived MPs (forward scatter; events <0.75 microm size), and DeltaPsi(m) (TMRM fluorescence). PS exposure was increased on resting and activated BSS platelets, and this was independent of the platelet size. MP formation by BSS platelets was generally enhanced. Cell shrinkage occurred on activation to form smaller, PS-exposing platelets in BSS and controls. A proportion of PS-exposing BSS and control platelets exhibited DeltaPsi(m) loss, but unlike controls, there was also loss of DeltaPsi(m) in the BSS platelets not exposing PS. Thus, BSS platelets undergo apoptotic-like events upon activation, with PS exposure and MP formation being enhanced. These events may play a role in the shortened survival in BSS, as well as affecting thrombin generation.
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PMID:Phosphatidylserine exposure and other apoptotic-like events in Bernard-Soulier syndrome platelets. 2065 88

Binding of Factor VIII to phosphatidylserine (PS)-expressing platelets is a key process in the intravascular pathway of the blood coagulation cascade. Activated by thrombin, FVIIIa acts as a cofactor on the surface of platelets. It is under debate whether and how annexin A5 influences FVIIIa binding to platelets. Here, we investigate FVIII binding to PS-containing vesicles as model platelets and its interplay with annexin A5 in buffer using fluorescence correlation spectroscopy (FCS). We find that activated FVIIIa, in contrast to inactivated FVIII, exhibits a striking binding anomaly as a function of PS content, marked by a sharp maximum of the binding constant around 11% PS, which is close to the natural PS content of platelets. Furthermore, we show that the addition of annexin A5 can both increase or decrease this FVIIIa binding depending on whether the relative PS content is lower or higher than the maximum binding value. We demonstrate in theory that the observed binding diagram supports the hypothesis that annexin shields PS, indicating a possible indirect regulatory role of annexin A5 in blood coagulation. The overall PS- and annexin-dependent binding behavior of activated FVIIIa is preserved in experiments in blood plasma, confirming the validity of our results under more physiological conditions.
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PMID:FVIII binding to PS membranes differs in the activated and non-activated form and can be shielded by annexin A5. 2195 89

The article presents results of observation of generation of thrombin in patients with ischemic heart disease in different terms after transcutaneous coronary intervention. The sampling included 37 patients with stable ischemic heart disease. The control group included 30 healthy individuals. To study system of hemostasis of this category of patients the test of generation of thrombin and its modification with added thrombomodulin were applied for evaluating anti-coagulant activity of system of protein C. The comparison of indicators of test of generation of thrombin in patients with ischemic heart disease before operation and in individuals of control group revealed no reliable differences (p > 0.05). The observation of patients with stable ischemic heart disease in various time-frame after mechanical re-vascularization of myocardium established significant increasing of generation of thrombin and decreasing of anticoagulant activity of system of protein C at 1-3 day after operation (p < 0.05). The positive correlation was established between endogenous thrombin potential and annexin 5, an early marker of dysfunction of endothelium in mentioned time-frame after operation (p = 0.0008; r = 0.57). The significant increasing of content of anti-inflammatory markers of C-reactive protein and fibrinogen was observed at 1-3 day after transcutaneous coronary intervention (p < 0.05). In that way, the study data give evidence to hyper-coagulation in patients with stable ischemic heart disease in early time-frame after operation despite normal values of activated partial thromboplastin time, prothrombin time, D-dimer and applied standard disaggregant therapy.
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PMID:[THE TEST OF GENERATION OF THROMBIN IN DYNAMICS IN PATIENTS AFTER TRANSCUTANEOUS CORONARY INTERVENTION]. 2618 89