Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.6 (thromboplastin)
 13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A simple assay method for platelet factor 4 is described. When factor Xa was added to a system containing antithrombin III in excess and heparin in low concentration, the amount of factor Xa immediately inactivated was found to be a function of the concentration of heparin. When an antiheparin such as platelet factor 4 was added, an increase of the residual activity of factor Xa was observed. The magnitude of this increase was shown to be correlated to the amount of heparin inactivation in the system. Platelet factor 4 could be assayed when the concentrations of antithrombin III, heparin, and factor Xa were maintained at a constant level and in excess. As an indicator of the reaction, factor Xa was measured with the chromogenic substrate benzoyl-Ile-Gou-Gly-Arg-p-nitroanilide (S-2222).
 ...
PMID:A simplified assay method for platelet factor 4 in plasma and in platelets with a chromogenic substrate. 72 Sep 57

The interaction of platelet factor four (PF-4) with glycosaminoglycans (GAG) was evaluated using fluorescence spectroscopy, a radioligand binding assay, and a functional assay utilizing antithrombin III and factor Xa. In these studies, we have (i) characterized the binding parameters for PF-4 to several forms of heparin and to dextran sulfate; (ii) examined the structural features of these glycosaminoglycans which support PF-4 binding; and (iii) examined the effects of selective digestion of the carboxy terminus of PF-4 on binding. The binding of PF-4 to unfractionated porcine intestinal mucosal heparin ([Mr] = 11,000) was specific and saturable, with a molar stoichiometry of PF-4 to heparin of approximately 4:1 and an apparent estimated Kd of 3 X 10(-8) M. Heparin fractions ([Mr] = 6,000) with either low or high affinity for antithrombin III bound to PF-4 with a similar apparent Kd. PF-4 also bound to dextran sulfate ([Mr] = 22,500) with an estimated apparent Kd of 6 X 10(-8) M and a molar stoichiometry of approximately 16:1. Carboxypeptidase Y (CP-Y) digestion of PF-4 progressively decreased GAG binding. After 30 min of digestion, by which time all of the carboxyterminal serine and glutamate, both of the two leucines, and approximately one-quarter of the four lysines were removed, the IC50 for heparin binding shifted from 10 to 150 nM. These studies demonstrated the effect of GAG polymer size and degree of sulfation on the affinity and stoichiometry of PF-4 binding, and the critical importance of the carboxy-terminal amino acids of PF-4 for binding to natural and synthetic GAGs.
 ...
PMID:The interaction of platelet factor four and glycosaminoglycans. 240 23

Murine monoclonal antibodies (mAb) were raised to a purified product of bovine PF-4, a 9,500 dalton protein with heparin neutralization activity comparable to that of human PF-4. Using a non-radioactive slide immunoenzymatic assay, four major classes of mAb could be identified when comparisons were made between purified antigens of PF-4 and beta-TG-like protein from both bovine and human species. Type 1 cross-reacted with all four antigens; type 2 reacted with PF-4s; type 3 reacted with only bovine PF-4 and beta-TG-like protein; and type 4 reacted only with bovine PF-4. Differences in immunoreactivities of types 1, 2 and 3 were retained throughout the growth of succeeding clones and in ascitic fluids. Using a modified factor Xa, S-2222 chromogenic substrate-heparin inhibition assay, no mAb was found to block PF-4's ability to neutralize heparin. mAbs representative of types 1, 2 and 3 were successfully raised in stable cell lines from at least second generation clones. These were purified with protein A agarose and found to be IgG1. By indirect immunocytofluorescence a purified type 2 mAb, 2E7, was found to specifically stain granules of human platelets and megakaryocytes, as well as masses (putative platelets within late stage megakaryocytes) without staining other cellular types in either bone marrow or peripheral blood. Species comparisons displayed positive staining for human, rat, and rabbit platelets and megakaryocytes, and negative staining for mouse, guinea pig and dog platelets and megakaryocytes. It seems likely that mAb, 2E7, is directed against an epitope, common to PF-4 of bovine, human, rabbit and rat.
 ...
PMID:Monoclonal antibodies to bovine platelet factor 4: species interactions to platelets and megakaryocytes using indirect immunocytofluorescence. 293 90

Bulk heparinized catheters (1 mm internal diameter) containing 10% heparin ionically bound, were tested in four human volunteers. Catheters containing 0% and 10% heparin were compared in each individual using ultrasound microflow velocimetry, permeability test, sequential determinations of activated partial thromboplastin time, heparin levels and generation of Fibrinopeptide A, beta thromboglobulin and Platelet factor 4. Although the release of heparin expressed by its anti-IIa activity is of similar range in the four individuals the release of anti-Xa activity is variable and generally of greater magnitude, suggesting a privileged migration of low molecular weight components of heparin. These antiproteasic activities of heparin are sufficient to inhibit fibrin formation and blood coagulation despite their relative inability to prevent platelet activation.
 ...
PMID:Thromboresistance of bulk heparinized catheters in human. 295 56

Platelet factor 4 is a polypeptide constituent of platelet alpha granules that is released during platelet aggregation and inhibits heparin-mediated reactions. Hageman factor (factor XII) is a plasma proenzyme that, when activated by certain negatively charged agents, initiates clotting via the intrinsic pathway of thrombin formation. In earlier studies using crude systems, platelet factor 4 inhibited activation of Hageman factor by dextran sulfate or cerebrosides, but not activation of Hageman factor by kaolin or ellagic acid. In the present study we examined the mechanisms of inhibition by platelet factor 4, using purified reagents. Platelet factor 4 inhibited activation of Hageman factor by ellagic acid, as measured by amidolysis of a synthetic substrate of activated Hageman factor, an effect inhibited by heparin or by an anti-platelet factor 4 antiserum. Coating glass tubes with platelet factor 4 before addition of normal plasma significantly lengthened the partial thromboplastin time of normal plasma. In addition, the clot-promoting properties of kaolin were inhibited by its prior exposure to platelet factor 4. Thus, the inhibitory properties of platelet factor 4 directed against the activation of Hageman factor were confirmed in a purified system. In this purified system, in contrast to earlier studies using crude systems, platelet factor 4 inhibited activation of Hageman factor by glass, ellagic acid, or kaolin.
 ...
PMID:Inhibition of the activation of Hageman factor (factor XII) by platelet factor 4. 304 34

Among extracellular biological processes the spatial control of blood clotting is a unique phenomenon. Localization in space has very important consequences in both normal and pathological conditions. Under physiological circumstances a clot is formed only in the vicinity of injury, albeit the prerequisites of coagulation are almost completely given in the whole circulation. The local character of blood clotting is secured by the following major conditions: The regulatory signal initiating coagulation-the damaged vascular wall-is itself a surface on which the majority of clotting reactions take place. The first enzyme, factor XII, of the intrinsic coagulation pathway is activated on the collagen fibers exposed in the damaged vascular wall, although the significance of this reaction in respect of the clotting process is ambiguous. On the membrane of platelets adhered to the damaged blood vessel is activated factor XI, too, which is a well-established participant of the intrinsic clotting process. The further consecutive reactions of coagulation are confined to the surface produced by injury, because the enzymes involved contain gamma-carboxyl-glutamyl side chains which are anchored through calcium bridges to the phospholipids of the platelet membrane. The last enzyme of the sequence is thrombin, which is released from the surface. The reactions taking place on the surface form an enzyme cascade, which amplifies the relatively weak triggering signal by several orders of magnitudes. Amplification is ensured not only by the enzyme-substrate relationship of the consecutive reaction partners, but also by spatial confinement, which endows the process with higher efficacy than could be expected on a statistical basis from reactions in solution. It contributes to the efficiency of enzyme cascade that the non-enzymatic regulatory proteins increase the activity of factors IXa and Xa, and thereby the overall process. While the partner of factor IXa, factor VIII, is captured from plasma, factor V, the partner of factor Xa, is derived from the platelets adhered to the damaged surface and orients the binding of factor Xa. The surface localization ensures the protection of the members of clotting system: In the activator complexes found on the surface, the spatial arrangement of clotting factors prevents the inactivation of factors by physiological inhibitors or by proteolytic enzymes and specific antibodies that appear in the circulation in pathological conditions. Platelet factor 4, derived from platelets, binds heparin and thereby markedly decreases the reactivity of antithrombin III, the physiological inhibitor of clotting factors. The above two circumstances are
 ...
PMID:Surface-governed molecular regulation of blood coagulation. 636 61

Platelet factor 4 (PF4) is a potent antiheparin in vitro. In view of the large amount of PF4 secreted from platelet alpha-granules during routine blood collection and processing techniques, the potential significance of this release was investigated using three measurements of heparin activity: the activated partial thromboplastin time (aPTT), the thrombin time, and factor Xa inactivation using the chromogenic substrate S2222 for assay of factor Xa. The results demonstrate that purified PF4 neutralizes heparin activity when added in increasing amounts to normal platelet-poor plasma containing a fixed concentration of commercial porcine gut mucosal heparin. This effect was seen when assaying heparin activity by all three methods. In addition, when heparin was added in increasing concentrations to pooled plasma samples that were collected from normal volunteers, there was neutralization of heparin activity in blood samples collected by routine citrate anticoagulation (CIT60) in comparison to blood samples collected simultaneously with platelet secretion inhibiting agents added to the anticoagulant (CIT+). This effect was seen when assaying heparin by the aPTT and thrombin time. These data confirm that both purified and secreted PF4 have significant antiheparin activity when heparin is added in vitro to normal plasma. Neutralization of circulating heparin by PF4 secreted during blood collection from anticoagulated patients could result in underestimation of the actual in vivo heparin concentration. In order to evaluate the significance of this effect, purified PF4 was added to plasma collected from heparinized patients and again PF4 neutralized heparin activity. This was seen, however, only when heparin activity was measured by the thrombin time or Xa inactivation assays. There was minimal shortening of the aPTT when PF4 was added in final concentrations up to 1000 ng/ml. When blood samples were simultaneously collected from anticoagulated patients by both routine and special collection methods, these results were confirmed. There was a significant difference between heparin activities measured in the CIT+ (secreted PF4 58 ng/ml) and CIT60 (secreted PF4 1074 ng/ml) plasma samples by both thrombin time and Xa inactivation. There was no difference, however, in the aPT when both types of plasma samples were simultaneously collected and assayed for each anticoagulated patient. This suggests that there may be circulating heparin fractions which can prolong the aPTT but which do not interact with PF4.(ABSTRACT TRUNCATED AT 400 WORDS)
 ...
PMID:The effect of platelet factor 4 (PF4) on assays of plasma heparin. 674 73

Eight human subjects were fed diets enriched in saturated fat (SF), or polyunsaturated fat (PUF) and after each dietary regimen the plasma heparinthrombin clotting time (HTCT) was determined. The HTCT of citrated plasma indicated reduced heparin-neutralizing activity (HNA) after PUF feeding compared with SF feeding. Platelet factor 4 (PF4) levels in the citrated plasma samples demonstrated an inverse correlation with the HTCT (r = 0.62). Experiments with purified PF4 indicated that the PF4 present in citrated plasma could only account for approximately 10% of the HNA. Plasma prepared in a manner which minimized in vitro release of platelet constituents contained significantly less PF4 after PUF feeding and indicated that most of the PF4 found in citrated plasma resulted from in vitro release. The factor Xa inhibitory activity of citrated plasma was not significantly altered by either of the dietary regimens.
 ...
PMID:Further observations on the effects of dietary fatty acid composition on platelet reactivity and blood coagulation in man and the influence of methodology on findings. 706 77

The purpose of this study was to investigate whether or not the systems of coagulation and fibrinolysis are activated after human recombinant erythropoietin therapy in patients with end-stage renal failure and renal anemia. Six thousand IU of human recombinant erythropoietin (EPOCH) were administered intravenously to 11 patients once a week for 8 weeks. Coagulation, fibrinolysis and platelet as well as renal functions were investigated before and after the EPOCH therapy. Platelet count did not increase in spite of improvement in anemia. No changes in prothrombin time, activated partial thromboplastin time, concentrations of fibrinogen, fibrinopeptide A, thrombin antithrombin III complex, fibrin/fibrinogen degradation products (FDP), FDP-E, FDP-D dimer, plasmin alpha 2-plasmin inhibitor complex were observed. Platelet factor 4 and beta-thromboglobulin also were unchanged. Reciprocal changes in serum creatinine concentrations over the duration of therapy were compared before and after therapy. There was no significant difference between the reciprocal changes in serum creatinine concentrations before and after therapy. The increases in hemoglobin did not correlate with the changes in coagulation, fibrinolysis and the other parameters, except for the change in prothrombin time. These results indicate that coagulation, fibrinolysis and platelet systems in end-stage renal failure patients were not affected by EPOCH administration, in spite of increase in hemoglobin.
 ...
PMID:Effects of recombinant human erythropoietin (EPOCH) on the coagulation and fibrinolytic systems and platelet function in pre-dialysis patients with chronic renal failure. 825 11

Platelet factor 4 (PF4), an abundant platelet alpha-granule protein, accelerates in vitro generation of activated protein C (APC) by soluble thrombin/thrombomodulin (TM) complexes up to 25-fold. To test the hypothesis that PF4 similarly stimulates endothelium-associated TM, we assessed the influence of human PF4 on thrombin-dependent APC generation by cultured endothelial monolayers. APC generated in the presence of 1 to 100 microg PF4 was up to 5-fold higher than baseline for human umbilical vein endothelial cells, 10-fold higher for microvascular endothelial cells, and unaltered for blood outgrowth endothelial cells. In an in vivo model, cynomolgus monkeys (n = 6, each serving as its own control) were infused with either PF4 (7.5 mg/kg) or vehicle buffer, then with human thrombin (1.0 microg/kg/min) for 10 minutes. Circulating APC levels (baseline 3 ng/mL) peaked at 10 minutes, when PF4-treated and vehicle-treated animals had APC levels of 67 +/- 5 ng/mL and 39 +/- 2 ng/mL, respectively (P <.001). The activated partial thromboplastin time (APTT; baseline, 28 seconds) increased maximally by 27 +/- 6 seconds in PF4-treated animals and by 9 +/- 1 seconds in control animals at 30 minutes (P <.001). PF4-dependent increases in circulating APC and APTT persisted more than 2-fold greater than that of controls from 10 through 120 minutes (P < or =.04). All APTT prolongations were essentially reversed by monoclonal antibody C3, which blocks APC activity. Thus, physiologically relevant concentrations of PF4 stimulate thrombin-dependent APC generation both in vitro by cultured endothelial cells and in vivo in a primate thrombin infusion model. These findings suggest that PF4 may play a previously unsuspected physiologic role in enhancing APC generation.
 ...
PMID:Platelet factor 4 enhances generation of activated protein C in vitro and in vivo. 1260 38


1 2 Next >>