Antiheparin Activity of Human Serum and Platelet Factor 4

1973 ◽  
Vol 30 (01) ◽  
pp. 093-105 ◽  
Author(s):  
C.H.J Sear ◽  
L Poller ◽  
F.R.C Path
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Blood Serum ◽  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Gel Filtration ◽  
Normal Serum ◽  
Platelet Factor ◽  
Mean Values ◽  
Antiheparin Activity

SummaryThe antiheparin activity of normal serum has been studied by comparing the antiheparin activities of sera obtained from normal whole blood, platelet-rich plasma and platelet-’free’ plasma with a purified platelet extract during differential isoelectric precipitation and by gel filtration chromatography.The mean values for the activity of PRP-serum and PFP-serum were 106% (S.D. 11) and 10% (S.D. 3) of untreated whole blood respectively. The activity of whole blood serum, PRP serum and whole blood serum plus platelet extract precipitated under identical physical conditions, i.e. pH 7.0, I =0.008, indicating that the activities of the three samples are probably associated with PF4. PF4 precipitated from human platelet extract at pH 4.0, but this is probably due to the difference in the two biochemical environments investigated, i.e. serum and platelet extract.The gel filtration experiments revealed striking similarities between the major antiheparin activities of serum and platelet extract. At physiological pH and ionic strength both activities were associated with high molecular weight material, but at physiological pH and elevated ionic strength both activities behaved as much smaller entities of molecular weight between 25,000 and 30,000 daltons and it seems very likely that both activities are associated with the same molecule, i.e. PF4.

The Effect of Lysed Platelets on Neutralization of Heparin In Vitro with Protamine as Measured by the Activated Coagulation Time (ACT)

1991 ◽  
Vol 66 (02) ◽  
pp. 213-217 ◽  
Author(s):  
Arthur P Bode ◽  
William J Castellani ◽  
Edna D Hodges ◽  
Susan Yelverton
Keyword(s):  
Platelet Rich Plasma ◽  
Coagulation Time ◽  
Platelet Factor ◽  
Platelet Suspension ◽  
Antiheparin Activity ◽  
Unit Increase ◽  
Heparin Anticoagulation ◽  
Heparin Activity ◽  
Activated Coagulation Time

SummaryThe effect of lysed platelets on the activated coagulation time (ACT) was studied in heparinized whole blood during titration with protamine. Frozen-thawed washed platelet suspension, or a chromatography fraction thereof, or autologous frozen-thawed platelet-rich plasma was added in various dilutions to freshly drawn blood anticoagulated with 3,000 USP units/1 heparin. After a 10 min incubation, the amount of protamine needed to restore the ACT to baseline ("protamine titration dose") was determined. We found that the protamine titration dose decreased in proportion to the amount of lysed platelet material added; expressed as a percentage of the total number of platelets present, each unit increase in lysed platelets produced a 1.7% ±0.8 (SD) reduction in the protamine dose needed to normalize the ACT. A heparin activity assay showed that this effect was not due to antiheparin activity of lysed platelets such as platelet factor 4 (PF4). Our data indicate that the procoagulant activity of platelet membranes reduced the sensitivity of the ACT to heparin. These findings suggest that membranous platelet microparticles may cause an inaccurate calculation, based on the ACT, of a protamine dose to reverse heparin anticoagulation in cardiopulmonary bypass procedures.

In Vivo Studies on the Inhibition of Coagulation by Fractionated Heparin and by a Heparin Analogue I. Effects of Heparin Fractions

1981 ◽  
Vol 46 (03) ◽  
pp. 612-616 ◽  
Author(s):  
U Schmitz-Huebner ◽  
L Balleisen ◽  
F Asbeck ◽  
J van de Loo
Keyword(s):  
Molecular Weight ◽  
Day Treatment ◽  
Gel Filtration ◽  
Weight Fraction ◽  
In Vivo Studies ◽  
Low Molecular Weight ◽  
High Molecular Weight Fraction ◽  
Platelet Factor ◽  
Heparin Fractions

SummaryHigh and low molecular weight heparin fractions obtained by gel filtration chromatography of sodium mucosal heparin were injected subcutaneously into six healthy volunteers and compared with the unfractionated substance in a cross-over trial. Equal doses of 5,000 U were administered twice daily over a period of three days and heparin activity was repeatedly controlled before and 2, 4, 8 hrs after injection by means of the APTT, the anti-Xa clotting test and a chromogenic substrate assay. In addition, the in vivo effect of subcutaneously administered fractionated heparin on platelet function was examined on three of the volunteers. The results show that s.c. injections of the low molecular weight fraction induced markedly higher anti-Xa activity than injections of the other preparations. At the same time, APTT results did not significantly differ. Unfractionated heparin and the high molecular weight fraction enhanced ADP-induced platelet aggregation and collagen-mediated MDA production, while the low molecular weight fraction hardly affected these assays, but potently inhibited thrombin-induced MDA production. All heparin preparations stimulated the release of platelet Factor 4 in plasma. During the three-day treatment periods, no side-effects and no significant changes in the response to heparin injections were detected.

Human High Molecular Weight Kininogen - A Secreted Platelet Coagulant Protein

1981 ◽  
Author(s):  
A H Schmaier ◽  
J Kuchibhotla ◽  
R W Colman
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Gel Filtration ◽  
Human Platelets ◽  
Metabolic Inhibitors ◽  
Ionophore A23187 ◽  
High Molecular Weight Kininogen ◽  
Contact Phase ◽  
Platelet Factor ◽  
Coagulant Activity

Platelets have been shown to contain a number of secret- able coagulant proteins, which participate as substrates or cofactors in plasma coagulation reactions. Since we have previously demonstrated that high molecular weight kininogen (HMWK) is immunochemically present in platelet extracts, we posited that HMWK is secreted during activation of platelets. Fresh normal platelets were washed by a combination of albumin-gradient and gel-filtration procedures. In 11 experiments the supernates of freeze-thaw lysates of normal human platelets contained a mean of 5.7 Units (range 3.16 to 8.14) of HMWK coagulant activity/3 × 1011 platelets. This coagulant activity was neutralized by a goat antiki- ninogen antibody. Using a 125I-HMWK tracer in PRP, the supernate of washed activated platelets contained 0.082% radioactivity as the starting PRP, suggesting that 14% of the total HMWK coagulant activity could be accounted for by plasma contamination. In four experiments, ionophore A23187 (15μM) induced a net secretion of 39% of the total platelet HMWK (range 16 to 49%). Platelet HMWK secretion by A23187 was concentration dependent (1 to 15 μM) . At A23187 (15μM) platelets released 75% 14C-5HT (range 61 to 99%) and 81% low affinity platelet Factor 4 (range 60 to 99%). Ninety-five percent of A23187-induced secretion of HMWK could be blocked by platelet pretreatment with metabolic inhibitors. LDH determinations indicated that only 5% (range 0 to 10%) of total secreted platelet HMWK could be attributed to lysis. Collagen and PGH2 also caused secretion of platelet HMWK coagulant activity. This study indicates that human platelets contain functional HMWK which may be secreted locally to modulate the reactions of the contact phase of plasma proteolysis.

Action of Erythrocyte Extract on the Prothrombin Consumption Test

1958 ◽  
Vol 194 (3) ◽  
pp. 527-530 ◽  
Author(s):  
Feliks Stanski ◽  
Armand J. Quick
Keyword(s):  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Normal Serum ◽  
Human Erythrocytes ◽  
Hemophilia B ◽  
Clotting Factor ◽  
Serum Factor ◽  
Rabbit Plasma

The prothrombin consumption time for human and rabbit whole blood and for platelet-rich plasma is approximately the same. The addition of an extract of human erythrocytes to the blood or plasma before clotting increases prothrombin consumption significantly except in platelet-poor rabbit plasma. If a mixture of aged normal serum and an extract of erythrocytes is added to the latter, the consumption of prothrombin is greatly increased. This potentiating activity of normal aged serum is also present in aged hemophilic serum, but is diminished in the serum from a patient with hemophilia B (PTC deficiency). The serum factor is destroyed at 60°C and is adsorbed with Ca3(PO4)2. The findings suggest that the cofactor of erythrocytin, the clotting factor in the erythrocyte, may be related to the agent lacking in hemophilia B.

scholarly journals Eġġ proteins in cod serum. Natural occurrence and induction by injections of oestradiol 3-benzoate

1971 ◽  
Vol 121 (5) ◽  
pp. 847-856 ◽  
Author(s):  
P. A. Plack ◽  
D. J. Pritchard ◽  
N. W. Fraser
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Double Diffusion ◽  
Normal Serum ◽  
Total Serum Protein ◽  
Total Serum ◽  
Natural Occurrence ◽  
Serum Samples ◽  
Male And Female ◽  
Oestradiol Benzoate

1. Before the uptake of water that precedes spawning, eggs of cod (Gadus morhua L.) contained 30% dry matter, of which 80% was protein. Some 75% of this protein was soluble in 0.5m-sodium chloride. The major components in the extract were two similar lipoproteins, of molecular weight about 400000, containing 21% lipid, some two-thirds of which was phospholipid, and about 0.5% protein phosphorus. 2. These lipoproteins were identified by immunochemical methods in the serum of female cod with developing ovaries, but not in the serum of male or of immature female fish. 3. The concentrations of egg proteins in the serum of female cod were determined by a serial-dilution double-diffusion immunological method, and were shown to increase with development of the ovaries, reaching a value of about 32mg/ml when the weight of the ovaries was 10% of the weight of the fish. 4. Immature male and female cod were injected intramuscularly with a solution of oestradiol-17β 3-benzoate in oil and the concentration of egg proteins in their serum was measured by the immunodiffusion method. The serum contained no detectable egg proteins before injection of the fish, but 30μg of oestradiol benzoate/kg gave rise to detectable amounts of egg proteins in 10 days, and with 300μg or 1mg of oestradiol benzoate/kg the concentration of egg proteins rose to 32mg/ml. The values for male and female cod were similar and represented about one-half of the total serum protein. 5. With a dose of 1mg of oestradiol benzoate/kg, egg proteins were first detected in the serum 2 days after injection and the concentration increased up to 10 days. 6. Serum samples taken before and 10 days after an injection of 1mg of oestradiol benzoate/kg were fractionated by gel-filtration on Sephadex G-200. The difference curves obtained from fractionation curves after and before injection confirmed the values of the concentrations of egg proteins obtained from the immunodiffusion test and showed that the concentrations of the normal serum components fell by 20–50% of the initial value, the high-molecular-weight globulins showing the most marked fall. 7. Egg proteins were detected in the liver and testes of the injected fish, but not in the ovaries.

Molecular and Functional Heterogeneity in Contaminants Isolated from Recalled Heparin. Impact on Anticoagulation and Potential Adverse Reactions

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4048-4048
Author(s):  
Jawed Fareed ◽  
Debra Hoppensteadt ◽  
Walter Jeske ◽  
Cafer Adiguzel ◽  
Omer Iqbal ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Adverse Reactions ◽  
Dermatan Sulfate ◽  
Platelet Factor 4 ◽  
Contact Activation ◽  
Clotting Time ◽  
Platelet Factor ◽  
Chromatographic Methods

Abstract The main contaminant in the recalled batches of unfractionated heparin (UFH) is reported to be oversulfated chondroitin sulfate (OSCS). No data on the composition and biologic properties of this reported OSCS is available. Moreover, it has been assumed that different batches of preparations contained similar forms of OSCS. Since this contaminant has been purposefully added to UFH and has been found in several batches from various suppliers and is also found in some of the low molecular weight heparins (LMWHs), it was hypothesized that some type of OSCS was used to supplement UFH preparations. To test this hypothesis four batches of contaminated UFH and two batches of LMWH were investigated. Four batches of UFH of the recalled products from the US suppliers and two batches of a LMWH from the European community were compared. The molecular weight profile studies were carried out using high performance liquid chromatographic methods. Each of these products were subjected to heparinase-1 digestion to determine the non-digestable components. The anticoagulant activities of each of these products were measured using the whole blood activated clotting time and thromboelastographic analysis. The effect of each product was also studied on thrombin generation (Fibrinopeptide A, thrombin antithrombin complex, and prothrombin F1.2) and contact activation. The anticoagulant assays (PT, APTT, TT, Heptest, ecarin clotting time), antiprotease profile (anti-Xa and anti-IIa) and thrombin generation inhibitory studies were carried out in the normal human plasma. Protamine and platelet factor 4 neutralization were also carried out in plasma. Non-heparin contaminants were also isolated from each of the heparins by digestion of heparin followed by alcohol precipitation and ion exchange chromatographic methods. The isolated components were further purified by physicochemical methods. Each of these components were profiled for the molecular, structural and biologic activity. In addition each of the contaminants were further characterized in terms of their interactions with SERPINS (AT III and HCII), platelet factor 4 and analyzed for anticoagulant activity in whole blood and citrated plasma systems. The four contaminated UFHs (H1-H4) did not exhibit any major differences in the molecular weight profile (14.8–15.6 KDa). The USP potency of these products were also similar (158–170 U/mg). The anticoagulant actions were comparable in the different whole blood and global assays. However, the anti-Xa and anti-IIa ratios were found to exhibit some variations (0.93–1.24). Each product showed differences in the heparinase resistant component (14–30%). H1 and H3 products also contained significant amounts of dermatan sulfate. Each of the contaminants exhibited different neutralization profiles with PF4 and protamine sulfate. The LMWH products were comparable in all of the studies including the molecular weight profile and biologic actions, however, they showed differences in the heparinase-1 digestion profiles. Moreover, the molecular weight of the contaminant obtained from the LMWH was lower (12.8 vs. 14.1–16.8 KDa). The contaminants also exhibited differences in thrombin generation markers. The USP potency of the heparin contaminants varied from 28–46 U/mg whereas the contaminant from the LMWH exhibited a potency of 38–46 USP U/mg. These studies suggest that the contaminants isolated from recalled batches of heparin are heterogenous in nature and may originate from multiple sources. Moreover, the contaminants obtained form LMWHs may exhibit additional structural and biologic differences. Therefore, the wide variations observed in the adverse reactions with recalled heparins may be due to compositional variations in the contaminants.

Human and Bovine Plasminogen-Free Thrombin, Purified by Means of Gel Filtration and Ion Exchange Chromatography

1966 ◽  
Vol 15 (03/04) ◽  
pp. 501-510 ◽  
Author(s):  
W Berg ◽  
K Korsan-Bengtsen ◽  
J Ygge
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Large Scale ◽  
Gel Filtration ◽  
Trisodium Citrate ◽  
Exchange Chromatography ◽  
Simple Method ◽  
Commercial Preparation ◽  
Usual Manner ◽  
Low Ionic Strength

SummaryA simple method for preparation of plasminogen-free human and bovine thrombin is described.Crude thrombin was prepared in the usual manner from oxalated plasma by means of adsorption on BaSO4, elution with trisodium citrate and activating the eluate from BaSO4 with tissue thromboplastin.This crude thrombin was purified by means of gel-filtration and chromatography on CM-Sephadex A-50.The gel-filtration was performed on three types of Sephadex, G-75, G-50, and G--25. By means of Sephadex G-75 the thrombin was well separated from the main part of inert protein and this type of Sephadex was used for the purification in large-scale. Separation of thrombin from protein of higher molecular weight was also obtained with Sephadex G-50 but not with Sephadex G-25 indicating a molecular weight of thrombin between 4000 and 10,000.The importance of using an elution buffer of sufficient high ionic strength for gel-filtration is shown. A great deal of the thrombin was adsorbed to the Sephadex if the gel-filtration was performed at a too low ionic strength.The final preparation contained 30,000 NIH units of thrombin per mg tyrosin and no detectable plasminogen.The commercial preparation “Topostasine” was also purified in the same manner, but the plasminogen content in “Topostasine” was high and could not be completely separated from thrombin.

Dissociation of Factor VIII in the Presence of Proteolytic Inhibitors

1978 ◽  
Vol 40 (02) ◽  
pp. 316-325 ◽  
Author(s):  
Ira I Sussman ◽  
Harvey J Weiss
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Factor Viii ◽  
Direct Evidence ◽  
Gel Filtration ◽  
High Ionic Strength ◽  
Procoagulant Activity ◽  
Low Molecular Weight ◽  
Bean Trypsin Inhibitor ◽  
Benzamidine Hydrochloride

SummaryWhen gel filtration of factor VIII is performed with buffers of high ionic strength (1.0 M NaCl or 0.25 M CaCl2), the procoagulant activity elutes with proteins of relatively low molecular weight. It has been suggested that in the presence of proteolytic inhibitors, the procoagulant activity would appear at the void volume. To test this hypothesis, chromatography with buffers of high ionic strength was performed in the presence of benzamidine hydrochloride, soy bean trypsin inhibitor, heparin, DFP, and aprotinin. Under all of these conditions, the procoagulant activity continued to elute with proteins of low molecular weight. Similar findings were obtained after chromatographing cryoprecipitate prepared from the plasma of a normal subject who had received heparin. Thus, at present there is no direct evidence to suggest that proteolysis is involved in the dissociation of factor VIII by buffers of high ionic strength.

Dissociation and Reassociation of Poliovirus II. Protein Components Obtained by Urea Treatment of the Virus Particle

1977 ◽  
Vol 32 (7-8) ◽  
pp. 632-636 ◽  
Author(s):  
Ursula Yamaguchi-Koll ◽  
K. J. Wiegers ◽  
R. Drzeniek
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Virus Particle ◽  
Gel Electrophoresis ◽  
Phosphate Buffer ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Urea Treatment ◽  
Primary Products ◽  
Protein Components

Abstract Dissociation of poliovirus by 9 ᴍ urea in 0.015 ᴍ NaCl at 25 °C resulted in the liberation of 35S RNA and of polypeptides sedimenting at 2S in sucrose gradients containing 9 ᴍ urea. However. a ribonucleopolypeptide (RNPP) complex sedimenting at 45S and oligomers of the viral polypeptides sedimenting at 7 -8S were found in addition to the monomers sedimenting at 2S when the urea concentration was lowered to 5 M after the dissociation procedure. Ribonuclease treatment prevents the appearance of the RNPP-complex. The amount of the RNPP-complex de­ creased, when the dissociation was performed at higher ionic strength. Under these conditions small amounts of empty capsids were detected. Polyacrylamide gel electrophoresis showed that the RNPP-complex contained the polypeptide VP1. The oligomers (7 -8S) contained the polypeptide VP3 and small amounts of VP2. The bulk of VP2 and some VP3 were found in the 2S position together with VP4. The molecular weight of the dissociation products in urea and phosphate buffer was determined by gel filtration to be about 30,000 for the monomeric polypeptides containing predominantly VP2 and about 70,000 for the oligomeric polypeptides containing predominantly VP3. Our results demonstrate that the oligomers and the RNPP-complex are not primary products obtained by dissociation of the virus particle by urea but are due to a reassociation of the poly­ peptides or of VP1 and RNA.

scholarly journals Gel Filtration Studies of Serum B12 Binding: An Abnormal Pattern in Patients with Vitamin B12 Deficiency

Blood ◽  
1969 ◽  
Vol 34 (6) ◽  
pp. 774-781 ◽  
Author(s):  
CHRISTINE LAWRENCE
Keyword(s):  
Molecular Weight ◽  
Vitamin B12 ◽  
Binding Capacity ◽  
Gel Filtration ◽  
Vitamin B12 Deficiency ◽  
Normal Serum ◽  
B12 Deficiency ◽  
Abnormal Pattern ◽  
Normal Controls

Abstract 57CoB12 was added to serum in vitro to study its binding by the three known serum B12-binders in patients with vitamin B12 deficiency and in normal controls. Gel filtration through columns of Sephadex G-200 was used to separate the low (beta) and high (alpha1 and beta) molecular weight B12-binding fractions. Electrophoresis on filter paper was used to separate the alpha1- and beta-globulins. The alpha1-globulin fraction in the serum of B12-deficient patients bound more of the added 57CoB12 than did this fraction in normal serum, presumably because this binder of the serum endogenous vitamin B12 is much less saturated in B12-deficiency. However, the total B12 binding capacity of the alpha1-globulin (for endogenous plus added vitamin B12) was lower in B12-deficient than in normal serum. The low molecular weight beta-binder bound more added 57CoB12 in B12-deficient than in normal serum, whereas the high molecular weight beta binder had a much lower B12-binding capacity in deficient than in normal serum. These abnormalities were independent of the cause of the vitamin B12 deficiency and disappeared after successful treatment with vitamin B12.

Sign in / Sign up

Export Citation Format

Share Document