Stypven Time Measurement of Plasma After Filtration: Effects of Cigarette Smoking

1979 ◽  
Author(s):  
F.C. Chao ◽  
J.L. Tullis ◽  
C.A. Alper ◽  
J. E. Silbert
Keyword(s):  
Cigarette Smoking ◽  
Platelet Rich Plasma ◽  
Membrane Vesicles ◽  
Time Measurement ◽  
Factor V ◽  
Differential Centrifugation ◽  
Healthy Male ◽  
Platelet Factor ◽  
Affect Factor ◽  
Millipore Filters

Normal plasma contains nonsedlmentable platelet factor-3 (NS-PF3) activity, thought to be caused by circulating platelet membrane fragments. Stypven time (ST), an assay for PF-3 activity, of plasma prepared by differential centrifugation and by filtration through 0.22 μ Millipore filters were Investigated. The average ST for platelet-rich plasma (PRP), low-spin platelet-poor plasma (LSPPP), medium-spin PPP (MSPPP), high-spin PPP (HSPPP) and filtered PPP (FPPP) was 28.0, 40.4, 43.4, 61.7 and 65.5 sec, respectively (27 determinations). Filtration of plasma did not affect factor V and X activities. Material eluted from filters after filtration of plasma consisted of membrane vesicles with high PF-3 activity. ST were then measured in plasma preparations obtained from smoking (S) (>15 cigarettes/day) and nonsmoking (NS) healthy male individuals, ages (A) between 45-64. Data obtained were grouped according to age and smoking habits (Gr. I, 9 S, A 45-64; Gr. II, 14 NS, A 45-54; Gr. III, 7 S, A 55-64; Gr. IV, 14 S, A 55-64) and subjected to two-way analysis variance employing the BMDP2V program. Significant shortening of ST was noted in LSPPP (p<0.02) and MSPPP (p<0.02) in smoking groups which, however, showed no significant differences in PRP (p>0.8), HSPPP (p>0.06) and FPPP (p>0.4). Results suggest smoking individuals exhibit significantly higher NS-PF3 activity in plasma.

Effect Of Prostacyclin (PGI2) On Human Platelet Aggregation And Secretion

1981 ◽  
Author(s):  
C M Chesney ◽  
D D Pifer
Keyword(s):  
Platelet Aggregation ◽  
Time Course ◽  
Platelet Rich Plasma ◽  
Dense Granule ◽  
Ionophore A23187 ◽  
Factor V ◽  
Platelet Factor ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Data Representative

PGI2,which increases platelet cAMP(Prostaglandins 13: 389,1977),is a potent inhibitor of aggregation and secretion .We stidued the time course of the same return of platelet function after exposure of platelets to PGI2.Sepharose 2B columns were equilibrated with Tyrode’s albumin buffer, pH7.5 (no Ca2+) containing PGI2 (534nM). Platelet rich plasma was applied and eluted with the same buffer. The filtered platelets(GFP) were then subsampled hourly after elution from the column. Fibrinogen was added to finel concentration of 1.7mg/ml. Platelet aggregation(PA) and release of 14C serotonin (5HT),platelet factor 4(PF4), and factor V (FV) were assayed after stimulation of the platelet by collagen(C), ADP,epinephrine(E), arachidonic acid(AA) and ionophore A23187(I). Data representative of 5 separate studies follow.I(20μg/ml) induced PA was 76%(Ohr),52%(1hr) and 61%(2hr and beyond). Release of 5HT, FV,and PF4 were 60%,1.89u,and 7.97 yg/10 pit, respectively, at time 0 and increased progressively, reaching a plateau at 2 hr. AA(500μg/ml) was 10%(0hr),30%(2hr),68%(3hr) and 8%(4hr). Release of 5HT paralleled PA but release of FV and PF4 remained suppressed for 4 hrs. In contrast α-granule (PF4 and FV)release by C(μg/ml)increased as PA increased while dense granule secretion remained suppressed. PA as well as a and dense granule secretion by ADP (10μM) were minimal during 4 hrs. PA and FV secretion by E (55μM) also remain inhibited for 4 hrs. In spite of this normal dense granule release occurred initially and declined progressively over 4 hours.

Alteration In Platelet Factor 3 Activity In Plasma In Association With Cigarette Smoking

1981 ◽  
Author(s):  
F C Chao ◽  
D M Kenney ◽  
J L Tullis ◽  
C A Alper ◽  
J E Silbert
Keyword(s):  
Cigarette Smoking ◽  
Age Groups ◽  
Membrane Vesicles ◽  
Serum Levels ◽  
Platelet Factor ◽  
Lag Period ◽  
The Difference ◽  
Properdin Factor B ◽  
Age Range

Changes in blood coagulation and platelet functions in vivo in healthy smoking and non-smoking individuals of different age groups were studied. Blood samples were obtained on four different occasions (6 months apart during 1978-1980) from each of the 21 smokers and 42 non-smokers (age range 35-79), and analyzed. Statistically significant changes (p < 0.03) associated with cigarette smoking are: 1) increases in platelet count and fibrinogen in plasma; 2) elevation in a platelet procoagulant, platelet factor-3 (PF-3) activity in platelet-poor plasma (PPP); 3) increases in serum levels of α1-antitrypsin, orosomucoid, haptoglobin and properdin factor B; and 4) shortening of the lag period of collagen-induced platelet aggregation. Filtration through Millipore filters removed membrane vesicles which are enriched with PF-3 activity from the PPP. The difference in PF-3 activity in filtered plasma between the smoking and non-smoking groups were no longer statistically significant. The results are consistent with the interpretation that enhanced PF-3 activity in plasma occurs in association with cigarette smoking and results from the liberation into plasma of platelet membranes enriched in PF-3 activity.

Platelet Factor 3 Availability During Platelet Clumping - Studied with a Method Based on a Chromogenic Peptide Substrate

1979 ◽  
Author(s):  
K Korsan-Bengtsen ◽  
B Christenson ◽  
I Andersson ◽  
M Johnsen
Keyword(s):  
Lipid Emulsion ◽  
Platelet Rich Plasma ◽  
Factor V ◽  
Factor X ◽  
Peptide Substrate ◽  
Platelet Factor ◽  
Normal Platelet ◽  
Per Se ◽  
Aggregation Of Platelets ◽  
Thrombin Formation

Platelet factor 3 (Pf3) together with factor V accelerates the conversion of prothrombin to thrombin by factor X a. Our method to determine platelet factor 3 activity is based on the measurement of thrombin formation with the substrate H-D-Phe-Pip-Arg-pNA in a system containing normal platelet poor plasma and Russells’ viper venom. There is a linear relationship between the rate of thrombin formation and the concentration of Pf3. The method is very sensitive. Thus 10 μl of frozen platelet rich plasma (PRP) or a lipid emulsion prepared from brain give absorbances > 2.0. When PRP is aggregated with ADP or collagen in concentrations which give maximal aggregation, no Pf3 activity whatsoever can be demonstrated. Treatment with kaolin or simply prolonged shaking of the PRP on the other hand increase the Pf3 activity extensively. Our conclusion is that aggregation of platelets per se do not make Pf3 available.

Measurement Of Platelet Factor 3 Availability By Using Chromogenic Substrate

1981 ◽  
Author(s):  
J Hársfalvi ◽  
J Chmielewska ◽  
Z S Latallo ◽  
L Muszbek
Keyword(s):  
Platelet Rich Plasma ◽  
Factor Xa ◽  
Limiting Factor ◽  
Factor V ◽  
Chromogenic Substrate ◽  
Platelet Factor ◽  
Activated Platelets ◽  
Diagnostic Potential ◽  
Photometric Technique ◽  
Rate Limiting

The measurement of platelet factor 3 (Pf 3) availability (the prothrombin converting procoagulant activity of platelet membran phospholipids) induced by collagen, ristocetin, arachidonic acid or other activating agents is a potentially valuable test in the diagnosis of platelet disorders. Its diagnostic potential, however is greatly impaired by the fact that RW clotting time method usually used for Pf 3 determination is influenced by many factors, its reproducibility is rather poor, and results are difficult to express in quantitative well defined biochemical terms.In the present paper a new method measuring Pf 3 activity by using a chromogenic substrate was developed. Platelet rich plasma(PRP) was incubated by various activating agents, then prothrombin thrombin conversion was started by the addition of purified factor Xa. After a certain interval the reaction was stopped by soybean trypsin inhibitor and thrombin formed during the reaction was quantified by the chromogenic substrate S-2238 (Kabi). By varying incubation time, prothrombin, factor V concentrations, the optimal conditions, where platelet phospholipid was the rate limiting factor, were determined. In these conditions Pf 3 activity was a linear function of the number of activated platelets. The results are expressed as the ratio of thrombin generated in activated and unactivated PRP. The method is a rather simple quantitative photometric technique, has an improved reproducibility, and results showing the increase in thrombin generation have well defined biochemical meaning.

Platelet Factor 3 Availability During Platelet Clumping - Studied with a Method Based on a Chromogenic Peptide Substrate

1979 ◽  
Author(s):  
K. Korsan-Bengtsen ◽  
B. Christenson ◽  
I. Andersson ◽  
M. Johnsen
Keyword(s):  
Lipid Emulsion ◽  
Platelet Rich Plasma ◽  
Factor V ◽  
Factor X ◽  
Peptide Substrate ◽  
Platelet Factor ◽  
Normal Platelet ◽  
Per Se ◽  
Aggregation Of Platelets ◽  
Thrombin Formation

Platelet factor 3 (Pf3) together with factor V accelerates the conversion of prothrombin to thrombin by factor X a. Our method to determine platelet factor 3 activity is based on the measurement of thrombin formation with the substrate H-D-Phe-Pip-Arg-pNA in a system containing normal platelet poor plasma and Russells’ viper venom. There is a linear relationship between the rate of thrombin formation and the concentration of Pf3. The method is very sensitive. Thus 10 μl of frozen platelet rich plasma(PRP) or a lipid emulsion prepared from brain give absor-bances > 2.0. When PRP is aggregated with ADP or collagen in concentrations which give maximal aggregation, no Pf3 activity whatsoever can be demonstrated. Treatment with kaolin or simply prolonged shaking of the PRP on the other hand increase the Pf3 activity extensively. Our conclusion is that aggregation of platelets per se do not make Pf3 available.

Residual platelet factor V ensures thrombin generation in patients with severe congenital factor V deficiency and mild bleeding symptoms

Blood ◽  
2010 ◽  
Vol 115 (4) ◽  
pp. 879-886 ◽  
Author(s):  
Connie Duckers ◽  
Paolo Simioni ◽  
Luca Spiezia ◽  
Claudia Radu ◽  
Paolo Dabrilli ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Coagulation Factor ◽  
Factor V ◽  
Platelet Factor ◽  
Factor V Deficiency ◽  
Undetectable Plasma ◽  
Fatal Bleeding ◽  
Tissue Factor Pathway

Abstract Coagulation factor V (FV), present in plasma and platelets, is indispensable to thrombin formation, yet patients with undetectable plasma FV seldom experience major bleeding. We used thrombin generation assays to explore the role of platelet FV in 4 patients with severe congenital FV deficiency (3 with plasma FV clotting activity [FV:C] < 1%). When triggered with tissue factor (TF) concentrations up to 50pM, platelet-poor plasma (PPP) from the patients with undetectable plasma FV showed no thrombin generation, whereas platelet-rich plasma (PRP) formed thrombin already at 1 to 5pM of TF. Thrombin generation in PRP from the FV-deficient patients was enhanced to near-normal levels by platelet activators (collagen or Ca2+-ionophore) and could be completely suppressed by specific FV inhibitors, suggesting FV dependence. Accordingly, platelet FV antigen and activity were measurable in all FV-deficient patients and platelet FVa could be visualized by Western blotting. Normalization of the tissue factor pathway inhibitor (TFPI) level, which is physiologically low in FV-deficient plasma, almost completely abolished thrombin generation in PRP from the FV-deficient patients. In conclusion, patients with undetectable plasma FV may contain functional FV in their platelets. In combination with low TFPI level, residual platelet FV allows sufficient thrombin generation to rescue these patients from fatal bleeding.

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.

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 Collagen Mediated Platelet Release on Plasma Prekallikrein Activation

1984 ◽  
Vol 51 (01) ◽  
pp. 037-041 ◽  
Author(s):  
K M Weerasinghe ◽  
M F Scully ◽  
V V Kakkar
Keyword(s):  
Platelet Aggregation ◽  
Healthy Volunteers ◽  
Platelet Rich Plasma ◽  
Age Groups ◽  
Inhibitory Effect ◽  
Age Group ◽  
Platelet Factor ◽  
Activated Platelets ◽  
Platelet Release ◽  
Collagen Treatment

SummaryCollagen mediated platelet aggregation caused -5.6 ± 6.7% inhibition and +39.1 ± 15.2% potentiation of prekallikrein activation in plasma from normal healthy volunteers between 20–40 and 50–65 years of age, respectively (n = 15, p <0.01). The amouns of platelet factor-four (PF4) released in the two groups were not significantly different. Collagen treatment in the presence of indomethacin caused +11.5 ± 3.6% and +59.6 ± 19.5% potentiation in the 20–40 and 50–65 age groups respectively (p <0.02). Adrenaline mediated platelet aggregation caused -55.2 ± 7.1% and -35.2 ± 8.3% inhibition in the 20–40 and 50–65 age groups, respectively. Collagen treatment of platelet-deficient-plasma and platelet-rich-plasma in EDTA also caused potentiation of prekallikrein activation.The results indicate that the observed degree of prekallikrein activation after platelet aggregation is a net result of the inhibitory effect of PF4 and the potentiatory effect of activated platelets. The potentiatory effect was greater after collagen treatment as compared to adrenaline treatment, and in the 50–65 age group as compared to the 20–40 age group.

Studies on the Pathogenesis of the Generalized Shwartzman Reaction

1964 ◽  
Vol 12 (02) ◽  
pp. 471-483 ◽  
Author(s):  
F Rodríguez-Erdmann
Keyword(s):  
Clotting Factor ◽  
Factor V ◽  
Clotting System ◽  
Platelet Factor ◽  
Trigger Mechanism ◽  
Haemorrhagic Diathesis ◽  
Shwartzman Reaction ◽  
Ca Ions

SummaryThe rôle of the clotting system in the pathogenesis of the generalized Shwartzman reaction (gSr) has been stressed in recent years. The clotting system is activated ubiquitously and as a result of it, fibrin is deposited intravascularly and a haemorrhagic diathesis develops. Evidence is presented herein, that endotoxin does not activate purified prothrombin, nor does endotoxin influence the convertion of prothrombin when it is activated in the presence of purified platelet-factor 3 (or caephalin) purified Ac-G (factor V) and Ca-ions.The trigger mechanism of the gSr also seems to be in the so-called prephase of clotting mechanism. Data are presented, which show that endotoxin activates the Hageman factor in vitro. The importance of this clotting factor and of platelet-factor 3 is discussed. Also the rôle played by the RES and cardiodynamic and vascular components are taken in consideration in the discussion.

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