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.

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.

The procoagulant effects of factor V Leiden may be balanced against decreased levels of factor V and do not reflect in vivo thrombin formation in newborns

2006 ◽  
Vol 95 (03) ◽  
pp. 434-440 ◽  
Author(s):  
Satu Hyytiäinen ◽  
Ulla Wartiovaara-Kautto ◽  
Veli-Matti Ulander ◽  
Risto Kaaja ◽  
Markku Heikinheimo ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Platelet Rich Plasma ◽  
Venous Blood ◽  
Factor V Leiden ◽  
Factor V ◽  
Cord Plasma ◽  
Adult Plasma ◽  
Thrombin Formation

SummaryThrombin regulation in newborns remains incompletely understood.We studied tissue factor-initiated thrombin formation in cord plasma in vitro, and the effects of Factor VLeiden (FVL) heterozygosity on thrombin regulation both in vitro and in vivo in newborns. Pregnant women with known thrombophilia (n=27) were enrolled in the study. Cord blood and venous blood at the age of 14 days were collected from 11 FVL heterozygous newborns (FVL-positive) and from 16 FVL-negative newborns. Prothrombin fragment F1+2 and coagulation factors were measured. Tissue factor-initiated thrombin formation was studied in cord platelet-poor plasma (PPP) of FVL-negative and -positive newborns, and in both PPP and platelet-rich plasma (PRP) of healthy controls. The endogenous thrombin potential (ETP) in cord PPP or PRP was ∼60% of that in adult plasma, while thrombin formation started ∼55% and ∼40% earlier in cord PPP and PRP, respectively. Further, in FVL-positive newborns thrombin formation started significantly earlier than in FVL-negative newborns. Exogenous activated protein C (APC) decreased ETP significantly more in cord than in adult PRP. In FVL-negative cord plasma 5nM APC decreased ETP by 17.4±3.5% (mean±SEM) compared with only 3.5±3.8% in FVL-positive cord plasma (p=0.01). FVL-positive newborns showed similar levels of F1+2 but significantly decreased levels of factor V compared with FVL negative newborns both in cord plasma (FV 0.82±0.07 U/ml vs. 0.98±0.05 U/ml, p=0.03) and at the age of two weeks (FV 1.15±0.04 U/ml vs. 1.32±0.05 U/ml, p=0.03). In conclusion, newborn plasma showed more rapid thrombin formation and enhanced sensitivity to APC compared with adult plasma. FVL conveyed APC resistance and a procoagulant effect in newborn plasma. Lack of elevated F1+2 levels in FVL-positive infants, however, suggested the existence of balancing mechanisms; one could be the observed lower level of factor V in FVL heterozygous newborns.

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.

scholarly journals von Willebrand factor binding to myosin assists in coagulation

2020 ◽  
Vol 4 (1) ◽  
pp. 174-180 ◽  
Author(s):  
Veronica H. Flood ◽  
Tricia L. Slobodianuk ◽  
Daniel Keesler ◽  
Hannah K. Lohmeier ◽  
Scot Fahs ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Factor V ◽  
Factor X ◽  
Skeletal Muscle Myosin ◽  
Myosin Binding ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract von Willebrand factor (VWF) binds to platelets and collagen as a means of facilitating coagulation at sites of injury. Recent evidence has shown that myosin can serve as a surface for thrombin generation and binds to activated factor V and factor X. We studied whether VWF can also bind myosin as a means of bringing factor VIII (FVIII) to sites of clot formation. A myosin-binding assay was developed using skeletal muscle myosin to measure VWF binding, and plasma-derived and recombinant VWF containing molecular disruptions at key VWF sites were tested. Competition assays were performed using anti-VWF antibodies. FVIII binding to myosin was measured using a chromogenic FVIII substrate. Thrombin generation was measured using a fluorogenic substrate with and without myosin. Wild-type recombinant VWF and human plasma VWF from healthy controls bound myosin, whereas plasma lacking VWF exhibited no detectable myosin binding. Binding was multimer dependent and blocked by anti-VWF A1 domain antibodies or A1 domain VWF variants. The specific residues involved in myosin binding were similar, but not identical, to those required for collagen IV binding. FVIII did not bind myosin directly, but FVIII activity was detected when VWF and FVIII were bound to myosin. Myosin enhanced thrombin generation in platelet-poor plasma, although no difference was detected with the addition of myosin to platelet-rich plasma. Myosin may help to facilitate delivery of FVIII to sites of injury and indirectly accelerate thrombin generation by providing a surface for VWF binding in the setting of trauma and myosin exposure.

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.

scholarly journals Thrombocytin, A Novel Platelet Activating Enzyme from Bothrops Atrox (MARAJOENSIS) Venom

1977 ◽  
Author(s):  
S. Niewiarowski ◽  
E.P. Kirby ◽  
G.J. Stewart ◽  
R. Turna ◽  
M. Wiedeman ◽  
...  
Keyword(s):  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Activate Factor ◽  
Factor X ◽  
Electron Microscopic ◽  
Platelet Factor ◽  
Small Vessels ◽  
Platelet Aggregates ◽  
Bothrops Atrox ◽  
Induced Aggregation

Thrombocytin (TCN) was purified from Bothrops atrox (BA) venom by precipitation with 1.2% Na-salicylate and chromatography on heparin-agarose column using increasing concentrations of lysine as eluent. It was homogeneous on SDS electrophoresis and had an apparent MW of 36,000. Immunoelectrophoresis with polyvalent anti-BA venom serum gave one cathodic arc indicating an isoelectric point higher than pH 8.6.TCN at a concentration of 1 yg/ml caused aggregation of human platelets, release of low affinity platelet factor 4 and serotonin, and stimulated platelets to retract fibrin.TCN was essentially free of fibrinogen clotting and fibrinolytic activities.TCN action on platelets was not mediated by the formation of thrombin since TCN did not activate Factor X or prothrombin and its action was not inhibited by hirudin.TCN is a serine protease since it was inhibited by DFP and it hydrolyzed a synthetic peptide, chromozyme UK (BZ-Val-Gly-Arg-pNA·HCl).TCN-induced aggregation of human platelets was completely inhibited by soy bean trypsin inhibitor, heparin, prostaglandin E1 and apyrase. Washed human platelets were 2-4 times less sensitive to TCN as compared to platelets in freshly prepared platelet rich plasma (PRP); their sensitivity to TCN gradually deteriorated during incubation of PRP at room temperature for 3 hours. Electron microscopic observations revealed formation of platelet aggregates characterized by pseudopod formation, centralization and partial loss of platelet granules. Infusion of TCN (3 yg) into the main artery of bat wing resulted in the formation of platelet aggregates seen on arterial and venous side which occasionally occluded small vessels.

scholarly journals Coinheritance of Factor V (FV) Leiden enhances thrombin formation and is associated with a mild bleeding phenotype in patients homozygous for the FVII 9726+5G>A (FVII Lazio) mutation

Blood ◽  
2003 ◽  
Vol 102 (12) ◽  
pp. 4014-4020 ◽  
Author(s):  
Elisabetta Castoldi ◽  
José W. P. Govers-Riemslag ◽  
Mirko Pinotti ◽  
Debora Bindini ◽  
Guido Tans ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Clinical Phenotype ◽  
Activated Protein C ◽  
Coagulation Factor ◽  
Factor Vii ◽  
Factor V ◽  
Factor X ◽  
Fv Leiden ◽  
Fvii Deficiency ◽  
Thrombin Formation

Abstract We investigated the role of thrombophilic mutations as possible modifiers of the clinical phenotype in severe factor VII (FVII) deficiency. Among 7 patients homozygous for a cross-reacting material-negative (CRM-) FVII defect (9726+5G&gt;A, FVII Lazio), the only asymptomatic individual carried FV Leiden. Differential modulation of FVII levels by intragenic polymorphisms was excluded by a FVII to factor X (FX) gene haplotype analysis. The coagulation efficiency in the FV Leiden carrier and a noncarrier was evaluated by measuring FXa, FVa, and thrombin generation after extrinsic activation of plasma in the absence and presence of activated protein C (APC). In both patients coagulation factor activation was much slower and resulted in significantly lower amounts of FXa and thrombin than in a normal control. However, more FXa and thrombin were formed in the plasma of the patient carrying FV Leiden than in the noncarrier, especially in the presence of APC. These results were confirmed in FV-FVII doubly deficient plasma reconstituted with purified normal FV or FV Leiden. The difference in thrombin generation between plasmas reconstituted with normal FV or FV Leiden gradually decreased at increasing FVII concentration. We conclude that coinheritance of FV Leiden increases thrombin formation and can improve the clinical phenotype in patients with severe FVII deficiency. (Blood. 2003;102:4014-4020)

scholarly journals Experimental Studies on Factor VIII Inhibitor Bypassing Activity

1977 ◽  
Author(s):  
H. Vinazzer
Keyword(s):  
Factor Viii ◽  
Calcium Chloride ◽  
Experimental Studies ◽  
Factor Xa ◽  
Factor Viii Inhibitor ◽  
Factor V ◽  
Factor X ◽  
Tissue Thromboplastin ◽  
Viii Inhibitor ◽  
Thrombin Formation

The exact action of factor VIII inhibitor bypassing activity (FEIBA) is still unclear. For this reason, a series of experimental studies was carried out. Procoagulant activities were examined by standard one-stage methods while factor Xa and thrombin were measured by chromogenic substrates. Activities of factors II, VII, IX, and X were similar to PPSB fractions. In addition, low factor V activity and a phospholipid were detected. No activated factor X was present in FEIBA but there was a trace amount of 2.1 NIH units of thrombin per 100 FEIBA units. On addition of calcium chloride slow thrombin formation could be observed which however, reached 1100 NIH units of thrombin per 100 FEIBA units within an incubation time of 10 min. The velocity of thrombin formation was greatly enhanced by addition of a PTT reagent and of thromboplastin respectively. Factor Xa on the other hand, was neither formed after addition of calcium chloride nor by a PTT reagent. Tissue thromboplastin however, activated Xa from FEIBA in the same manner as a PTT reagent plus barium sulfate plasma. From these results, the conclusion could be drawn that thrombin could readily be made available from FEIBA while activation of Xa either needed the complete endogenous pathway or the presence of tissue thromboplastin. The procoagulant activity of FEIBA therefore, could be attributed to direct thrombin formation. By this process, an activation of the clotting mechanism in plasmas deficient in endogenous coagulation factors, and a complete independence from the presence or absence of a specific antibody could be explained.

Levels of Platelet Factor 3 in Citrate Plasma and in Whole Blood as Determined by A Chromogenic Peptide Substrate Assay

1979 ◽  
Author(s):  
H. Sandberg ◽  
A.-K. Gellerbring ◽  
L.-O. Andersson
Keyword(s):  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Blood Sampling ◽  
Chromogenic Substrate ◽  
Plasma Samples ◽  
Peptide Substrate ◽  
Platelet Factor ◽  
No Release ◽  
Sensitive Method

A newly developed sensitive method for determination of platelet factor 3 (PF 3) using a chromogenic substrate (S 2238) (Sandberg and Andersson, Thromb. Res., in press) was used for comparison of the PF 3 levels in platelet-rich plasma (PRP) in citrate with the levels in PRP in the anticoagulant EDTA/citrate/PCE 1/theophylline. It was shown that in citrate PRP, release of PF 3 was started after about 20 minutes from blood sampling. Release of β-thromboglobulin (β-tg) was found to occur simultaneously with the release of PF 3. No release of PF 3 or β-tg could be detected within 3 hours from blood sampling in PRP in the EDTA/citrate/PCE 1/theophylline anticoagulant.The PF 3 level in whole blood was measured by a method which was a modification of the method used for plasma samples. It was found that, using the same donor, the PF 3 level of plasma and blood in the EDTA/citrate/PCE 1/theophylline anticoagulant was essentially the same. The levels of PF 3 found in blood upon standing in citrate or in EDTA/citrate/PCE 1/theophy11ine anticoagulant were in accordance with the levels found in plasma. Experiments with whole blood without any anticoagulant showed that release of PF 3 begin to occur simultaneously with release of β-tg, about 5 minutes before clotting. Thus the data show that PF 3 and βtg are released in parallel.

The Role of Phospholipids and Factor Va in the Mechanism of Prothrombin Activation

1979 ◽  
Author(s):  
J. Rosing ◽  
G. Tans ◽  
J.W.P. Govers-Riemslag ◽  
R.F.A. Zwaal ◽  
H.C. Hemker
Keyword(s):  
Kinetic Parameters ◽  
Factor Xa ◽  
Clotting Factor ◽  
Factor V ◽  
Factor X ◽  
Factor Va ◽  
Prothrombin Activation ◽  
Thrombin Formation

The kinetic parameters of the conversion of prothrombin into thrombin by activated clotting factor X (factor Xa) have been determined in the absence and presence of Ca2+, phospholipid (phosphatidyl serine/phosphatidylcholine vesicles) and activated blood clotting factor V (factor Va). In free solution the Km for prothrombin is 298 μM which is well above its plasma concentration of 4μM. Under these conditions the Vmax of thrombin formation is 1.25 Moles min-1 Mole Xa -1. When phospholipid is present the km for prothrombin drops to 0.1μM while the Vmax is only slightly affected (3 Moles min-1 Mo Le Xa -1). For the complete prothrombin activating complex consisting of factor Xa, factor Va, Ca2+ and phospholipids the kinetic constants greatly favour thrombin formation. A for prothrombin of 0.26μM and a Vmax of 2130 Moles min-1 Mole xa -1 are measured under these conditions. These results help to elucidate the role of phospholipid and factor Va in prothrombin activation. The earlier observed rate enhancements caused by phospholipid and factor Va are explained as effects on the Km for prothrombin and the Vmax of thrombin formation, respectively. The changes of the kinetic parameters for prothrombinase complexes of various composition will be considered with respect to the function of the accessory components in the mechanism of prothrombin activation. Implications of these data for in vivo blood coagulation will be discussed.

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