Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effect of phospholipase C (EC 3.1.4.3) on human blood platelets has been studied. Phospholipase C from Bacillus cereus was purified to homogeneity as judged by analytical and sodium dodecyl sulphate disc gel electrophoresis and by immunoelectrophoresis. Human platelets isolated from platelet-rich plasma by gel filtration or by centrifugation and washing were incubated with phospholipase C. A loss of 20-45% of the total platelet phospholipid was observed, whereas 88% was hydrolyzed when platelet homogenates were submitted to identical enzyme treatment. Intact platelets lost 50-75% phosphatidylethanolamine, 20-50% phosphatidylcholine, and 20-25% phosphatidylserine. Sphingomyelin was not a substrate for the enzyme under the conditions used. The platelets contained no detectable endogenous phospholipase C activity. The loss of phospholipid was not accompanied by aggregation of the platelets, nor did the platelets lose their ability to aggregate with ADP or thrombin. Total platelet factor 3 releasable by freezing and thawing was reduced. Measurements of releasable platelet factor 4 and the efflux of serotonin showed that no release reaction was triggered even when up to 45% of the total phospholipid in the platelets was hydrolyzed. When sphingomyelinase was added together with, before, or after phospholipase C, aggregation occurred. Sphingomyelinase alone gave no aggregation. The gel-filtered platelets also aggregated upon addition of purified phospholipase C from Clostridium perfringens. The distribution of phospholipids in the platelet membrane is discussed.
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PMID:The effect of phospholipase C on human blood platelets. 81 57

Platelet factor 4 (heparin neutralizing) activity shortens the thrombin time of a heparinized plasma. In the proposed procedure (I) a heparin thrombin time curve is constructed by adding gradually increasing amounts of heparin to a commercial plasma substrate and determining thrombin times, (2) a suitable concentration of heparin that gives highest reproducible thrombin time is selected and added to the substrate, (3) thrombin times are determined for the heparinized substrate before and after addition of a test material containing platelet factor 4. The two thrombin times are converted to heparin concentration by reference to the heparin thrombin time curve. The difference in heparin concentrations represents platelet factor 4 activity. When lyophilized commercial plasma is used as substrate larger quantities of heparin can be employed in the system, resulting in improved sensitivity and precision.
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PMID:An improved procedure for quantitation of platelet factor 4. 86 92

1. The distribution of the heparin-neutralizing factor (platelet factor 4, PF4) in subcellular organelles of blood platelets of rabbits and man was investigated. 2. In both species the organelles storing 5-hydroxytryptamine (5-HT storage organelles) contained only trivial amounts of PF4. 3. In contrast, the content of PF4 was highest in the subcellular fractions rich in alpha-granules. 4. In conclusion, PF4 is probably localized in the alpha-granules and therefore the platelets contain at least two types of organelles (5-HT organelles and alpha-granules) capable of releasing their contents in response to the same stimuli, such as exposure to collagen, thrombin, etc.
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PMID:Subcellular localization of the heparin-neutralizing factor in blood platelets. 95 Jun 2

Dialysis of blood and plasma was performed in vitro, in a 'mini-Kill' dialyser as well as in dialysis bags. A marked shortening of the thrombin-clotting time was observed, indicating fall in heparin anticoagulant effect. The concentration of heparin, however, as measured by polybrene titration, was substantially less reduced. Fibrin formation, as evidenced by the ethanol gelation test, occurred more often in the dialysed than in the control plasma. In conclusion, the discrepancy between concentration and anticoagulant effect of heparin could be partly explained by influx from the dialysate of calcium, magnesium, and acetate ions. The fibrin-polymerizing effect of these ions was confirmed by a shortening of the clotting time with Reptilase, a proteolytic enzyme not influenced by thrombin inhibitors such as heparin. In addition, liberation of platelet factor 4 may be responsible for some reduction in antithrombin activity of heparin. No evidence of heparin being dialysed or adhering to the dialysis membrane was found.
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PMID:Impaired anticoagulant effect of heparin in the artificial kidney. An experimental study. 100 46

Platelet factor 4 was isolated by gel filtration from the soluble release products of thrombin-aggregated washed human platelets as a proteoglycan-platelet factor 4 complex of molecular weight 358 000, Stokes radius (r-s) of 14.0 nm, sedimentation coefficient (s) of 7.1 S and frictional ratio (f/f-o) of 3.04. The complex was dissociated at high ionic strength (I equals 0.75) and the proteoglycan separated from platelet factor 4 by gel filtration. Platelet factor 4 had a molecular weight of 27 100, r-s of 2.52 nm, s of 2.4 S and f/f-o of 1.26, was insoluble under physiological conditions but readily soluble at pH 3. Under these conditions platelet factor 4 dissociated into four subunits with a molecular weight of 6900, r-s of 1.92 nm, s of 0.8 S, and f/f-o of 1.52. Qualitative N-terminal amino acid analysis showed the presence of glutamic acid or glutamine as the major end group. Platelet factor 4 was compared with protamine sulphate, which has similar biological properties, by electrophoresis at pH 2.2, in which both migrated as single bands but with differing mobility, and by amino acid analysis which showed a more normal distribution of residues than occurred in protamine sulphate. Of the basic amino acids platelet factor 4 (molecular weight 27 100) contained 5.97% arginine, 3.18% histidine, and 12.31% lysine compared to protamine sulphate with 64.2% arginine, 0.6% lysine and no histidine. A partial specific volume (v) of 0.747 was calculated for platelet factor 4 from its amino acid analysis. A membrane fraction with antiheparin activity, an isopycnic density of 1.090-1.110 and r-s of 15-35 nm, was also isolated by sucrose density gradient centrifugation from the ultrasonicated insoluble platelet residue remaining after thrombin-induced aggregation of washed human platelets. Trypsin treatment of the membrane fraction neither solubilised nor destroyed the activity.
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PMID:Platelet antiheparin activity. The isolation and characterisation of platelet factor 4 released from thrombin-aggregated washed human platelets and its dissociation into subunits and the isolation of membrane-bound antiheparin activity. 112 93

1. X537A at concentrations below 10 muM can liberate platelet serotonin from washed human platelets without inducing the platelet release reaction. Up to 100% of serotonin preabsorbed by the platelets can be liberated before initiation of the release reaction. 2. Concentrations of X537A above 10muM initiate the platelet release reaction, with a maximum release of adenine nucleotides and platelet factor 4 antigen comparable to that obtained with 1.25 units thrombin/ml. 3. The changes in ATP metabolism at the concentration necessary for X537A-induced release are more profound than those in platelets exposed to concentrations of thrombin or A23187 giving the same degree of release, and approach those seen with high concentrations of A23187. At concentrations where serotonin is liberated but no adenine nucleotide or platelet factor 4 antigen is released, short time incubation causes no change in the level of metabolic ATP.
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PMID:Metabolic aspects of the secretion of stored compounds from blood platelets. IV. Effects of ionophore X537A on washed platelets. 127 64

Some traditional coagulation assays and several new molecular markers of hemostatic activation were measured in 37 patients with spinal cord injury (SCI). Twenty one of the patients (57%) developed deep vein thrombosis (DVT). The radiofibrinogen uptake test (RFUT) was used to diagnose DVT. Thirty eight percent of quadriplegic and 88% of paraplegic patients developed DVT (p < 0.005). No significant differences were found in platelet counts, mean platelet volumes, fibrinogen levels, von Willebrand factor (Ag) levels, platelet factor 4 and beta thromboglobulin concentrations between the groups with and without DVT. Fibrinopeptide A, thrombin/antithrombin III (TAT) complexes and plasma D-dimer levels were significantly higher in the patients with thrombosis. Most patients with DVT had elevated TAT complex levels up to three days before the RFUT became positive. D-dimer levels were highest after the diagnosis had been made.
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PMID:Thrombosis in spinal cord injury. 129 Jan 64

Rhynchophylline (Rhy) inhibited rabbit platelet aggregation induced by arachidonic acid (AA), collagen, and ADP. The values of IC50 were 0.72, 0.74, and 0.67 mmol.L-1, respectively. Rhy reduced the thromboxane B2 (TXB2) generation in PRP induced by collagen but failed to reduce that induced by AA. Rhy suppressed malondialdehyde (MDA) formation in platelet suspension stimulated by thrombin, inhibited the platelet factor 4 (PF4) release. It did not alter intraplatelet cAMP concentration. Rhy 10-20 mg.kg-1 iv showed a significant inhibition of venous thrombosis and cerebral thrombosis in rats.
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PMID:Inhibitory effect of rhynchophylline on platelet aggregation and thrombosis. 131 85

Effects of zinc and calcium ions on the heparin-neutralizing abilities of histidine-rich glycoprotein (HRG) and platelet factor 4 (PF4) were examined. Both HRG and PF4 effectively neutralized the ability of heparin to accelerate the activated protein C (APC) and the thrombin inhibitions by protein C inhibitor (PCI). the heparin-neutralizing ability of HRG in the APC inhibition by PCI, however, was decreased in a Ca(2+)-dependent manner and apparently lost at 1 mM Ca2+, while it was enhanced by Zn2+ regardless of the presence or absence of Ca2+. The heparin-neutralizing ability of HRG in the thrombin inhibition by PCI was not affected by Ca2+. In contrast to HRG, there was no significant difference in the heparin-neutralizing ability of PF4 in the presence or absence of 1 mM Ca2+. These results strongly suggest additional physiological functions of HRG and PF4 as modulators of PCI.
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PMID:Modulation of protein C inhibitor activity by histidine-rich glycoprotein and platelet factor 4: role of zinc and calcium ions in the heparin-neutralizing ability of histidine-rich glycoprotein. 131 17

The ability of several low molecular weight (LMW) heparins and unfractionated heparin (UFH) to inhibit thrombin generation, and their anti-Xa and anti-IIa activities, were measured in the absence and presence of platelet factor 4 (PF4). The LMW heparins studied were 2-5 times less potent, on a weight basis, than UFH as inhibitors of thrombin generation in platelet-poor plasma; the inhibition of thrombin generation by LMW heparins correlated better with their anti-IIa activity (r = 0.98) than with their anti-Xa activity (r = 0.69). At low concentrations of PF4, the activity of LMW heparins in the thrombin generation test was neutralized less than that of UFH, but at higher PF4 concentrations all their activities could be neutralized except in anti-Xa assays. These observations support the hypothesis that anti-IIa activity is important for inhibition of thrombin generation by LMW heparins in vitro. However, when all the anti-IIa activity of LMW heparins was neutralized by PF4, considerable inhibitory activity remained in thrombin generation and anti-Xa assays, indicating that a portion of the anti-Xa activity of LMW heparins also contributes towards inhibition of thrombin generation.
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PMID:Inhibition of thrombin generation by heparin and low molecular weight (LMW) heparins in the absence and presence of platelet factor 4 (PF4). 132 21


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