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.

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.

Abstract 43: Prothrombinase Assembled with Factor V Leiden is Resistant to Inhibition by Tissue Factor Pathway Inhibitor α Lowering the Procoagulant Threshold for Initiation of Coagulation

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Jeremy P Wood ◽  
Lisa M Baumann Kreuziger ◽  
Susan A Maroney ◽  
Rodney M Camire ◽  
Alan E Mast
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Tissue Factor Pathway Inhibitor ◽  
Platelet Rich Plasma ◽  
Anticoagulant Activity ◽  
Factor V Leiden ◽  
Factor Xa ◽  
Factor V ◽  
Activation Threshold ◽  
Tissue Factor Pathway

Factor V (FV) assembles with factor Xa (FXa) into prothrombinase, the enzymatic complex that converts prothrombin to thrombin. Tissue factor pathway inhibitor α (TFPIα) inhibits prothrombinase by high affinity interactions with FXa-activated FV and the FXa active site, thereby blocking the initiation of coagulation. FV Leiden (FVL) is strongly linked to venous thrombosis through its resistance to degradation by activated protein C (aPC), which enhances the propagation of coagulation. FVL combined with a 50% reduction in TFPI causes severe thrombosis and perinatal lethality in mice, suggesting that FVL also promotes the initiation of coagulation. To examine this possibility, thrombin generation assays initiated with limiting FXa were performed with control or FVL plasma and platelet-rich plasma (PRP). The activation threshold for thrombin generation was 10 to 20 pM FXa in 10 control plasmas, but was 5 pM in 4 of 10 homozygous FVL plasmas. FVL PRP had a similar decrease in the activation threshold. The differences in activation threshold were totally normalized by an anti-TFPI antibody, while exogenous TFPIα and a FV-binding peptide that mimics TFPIα had reduced anticoagulant activity in FVL plasma, revealing that the procoagulant effects of FVL in these assays rely on TFPIα. Next, FVL plasmas were studied in fibrin clot formation assays, as they are sensitive to small amounts of thrombin. In reactions activated with 0.5 pM FXa, 1 of 8 control plasmas, compared to 7 of 8 homozygous FVL plasmas, clotted within 60 minutes, with differences again normalized by the anti-TFPI antibody. In prothrombinase activity assays using purified proteins, TFPIα was a 1.7-fold weaker inhibitor of prothrombinase assembled with FVL compared to FV. Thus, in addition to its aPC-mediated effect on the propagation of coagulation, FVL is resistant to TFPIα inhibition, exerting a procoagulant effect on coagulation initiation. This is evident in responses to small stimuli, where TFPIα blocks clotting in plasmas with FV but not FVL. The TFPIα-mediated modulation of the procoagulant threshold may explain the severe perinatal thrombosis in FVL mice with decreased TFPI and be clinically relevant in the clotting associated with oral contraceptives, which cause acquired TFPI deficiency.

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 The effect of platelets upon factor Xa-catalyzed activation of factor VII in vitro

Blood ◽  
1988 ◽  
Vol 72 (2) ◽  
pp. 396-401 ◽  
Author(s):  
LV Rao ◽  
SI Rapaport
Keyword(s):  
Surface Membrane ◽  
Calcium Ionophore ◽  
Factor Xa ◽  
Factor Vii ◽  
Ionophore A23187 ◽  
Exogenous Factor ◽  
Platelet Factor ◽  
Anionic Phospholipids ◽  
Factor Va ◽  
Activated Platelets

Abstract The authors have investigated the ability of platelets to enhance factor Xa-catalyzed activation of factor VII. Unstimulated platelets were without effect, whereas freeze/thawed platelets substantially enhanced activation. Antifactor V antibodies did not diminish the enhancement. Platelets activated by thrombin, collagen, or calcium ionophore A23187 also enhanced factor Xa-catalyzed activation of factor VII. In contrast to their lack of effect upon freeze/thawed platelets, antifactor V antibodies abolished augmented factor VII activation induced by activated platelets. Adding exogenous factor Va to unstimulated platelets failed to enhance factor Xa-catalyzed activation of factor VII, nor did adding exogenous factor Va to activated platelets augment activation beyond that observed with activated platelets alone. These observations can be interpreted as follows: (1) factor Va does not function as a cofactor for factor Xa-catalyzed activation of factor VII; (2) anionic phospholipids are a known cofactor for factor Xa-catalyzed activation of factor VII, and freeze/thawed platelets probably enhance activation by making anionic phospholipids on disrupted platelet membranes available to function as a cofactor; (3) the presumed binding of factor Xa to exogenous factor Va on unstimulated platelets is insufficient in itself to augment factor Xa-catalyzed activation of factor VII; (4) activated platelets augment factor Xa-catalyzed factor VII activation because activation allows both factor Xa to bind to released platelet factor V(a) and makes available a surface membrane component, probably anionic phospholipids, with which the bound factor Xa interacts.

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 The effect of platelets upon factor Xa-catalyzed activation of factor VII in vitro

Blood ◽  
1988 ◽  
Vol 72 (2) ◽  
pp. 396-401
Author(s):  
LV Rao ◽  
SI Rapaport
Keyword(s):  
Surface Membrane ◽  
Calcium Ionophore ◽  
Factor Xa ◽  
Factor Vii ◽  
Ionophore A23187 ◽  
Exogenous Factor ◽  
Platelet Factor ◽  
Anionic Phospholipids ◽  
Factor Va ◽  
Activated Platelets

The authors have investigated the ability of platelets to enhance factor Xa-catalyzed activation of factor VII. Unstimulated platelets were without effect, whereas freeze/thawed platelets substantially enhanced activation. Antifactor V antibodies did not diminish the enhancement. Platelets activated by thrombin, collagen, or calcium ionophore A23187 also enhanced factor Xa-catalyzed activation of factor VII. In contrast to their lack of effect upon freeze/thawed platelets, antifactor V antibodies abolished augmented factor VII activation induced by activated platelets. Adding exogenous factor Va to unstimulated platelets failed to enhance factor Xa-catalyzed activation of factor VII, nor did adding exogenous factor Va to activated platelets augment activation beyond that observed with activated platelets alone. These observations can be interpreted as follows: (1) factor Va does not function as a cofactor for factor Xa-catalyzed activation of factor VII; (2) anionic phospholipids are a known cofactor for factor Xa-catalyzed activation of factor VII, and freeze/thawed platelets probably enhance activation by making anionic phospholipids on disrupted platelet membranes available to function as a cofactor; (3) the presumed binding of factor Xa to exogenous factor Va on unstimulated platelets is insufficient in itself to augment factor Xa-catalyzed activation of factor VII; (4) activated platelets augment factor Xa-catalyzed factor VII activation because activation allows both factor Xa to bind to released platelet factor V(a) and makes available a surface membrane component, probably anionic phospholipids, with which the bound factor Xa interacts.

scholarly journals Deficiency of factor Xa-factor Va binding sites on the platelets of a patient with a bleeding disorder

Blood ◽  
1979 ◽  
Vol 54 (5) ◽  
pp. 1015-1022 ◽  
Author(s):  
JP Miletich ◽  
WH Kane ◽  
SL Hofmann ◽  
N Stanford ◽  
PW Majerus
Keyword(s):  
Binding Sites ◽  
Factor Xa ◽  
Coagulation Factors ◽  
Severe Bleeding ◽  
Factor V ◽  
Platelet Factor ◽  
Platelet Surface ◽  
Factor Va ◽  
Number Of Binding Sites ◽  
Stimulation Of

Factor V (Va) is essential for binding of factor Xa to the surface of platelets. After thrombin treatment, normal platelets release at least five times more factor Va activity than is required for maximal factor Xa binding. The concentration of factor V activity obtained after thrombin stimulation of 10(7) normal platelets is sufficient to allow half-maximal factor Xa binding to 10(8) platelets (10% normal, 90% factor-V deficient). Therefore, factor Va activity is not limiting in platelet-surface factor Xa binding and prothrombin activation in normal platelets; some other components limit the number of binding sites. We report studies of a patient (M.S.) with a moderate to severe bleeding abnormality whose platelets are deficient in the platelet-surface component required for the factor Va-factor Xa binding. The patient's platelet factor Va activity released after thrombin treatment is normal, but factor Xa binding is 20%-25% of control values at saturation. Abnormal prothrombin consumption in a patient with normal plasma coagulation factors and platelet function suggests a disorder in platelet-surface thrombin formation.

Surface expression and functional characterization of α-granule factor V in human platelets: effects of ionophore A23187, thrombin, collagen, and convulxin

Blood ◽  
2000 ◽  
Vol 95 (5) ◽  
pp. 1694-1702 ◽  
Author(s):  
L. Alberio ◽  
O. Safa ◽  
K. J. Clemetson ◽  
C. T. Esmon ◽  
G. L. Dale
Keyword(s):  
Functional Characterization ◽  
Factor Xa ◽  
Annexin V ◽  
Surface Expression ◽  
Human Platelets ◽  
Ionophore A23187 ◽  
Factor V ◽  
Glycoprotein Vi ◽  
Activated Platelets ◽  
Simultaneous Activation

Factor V (FV) present in platelet -granules has a significant but incompletely understood role in hemostasis. This report demonstrates that a fraction of platelets express very high levels of surface-bound, -granule FV on simultaneous activation with 2 agonists, thrombin and convulxin, an activator of the collagen receptor glycoprotein VI. This subpopulation of activated platelets represents 30.7% ± 4.7% of the total population and is referred to as convulxin and thrombin–induced-FV (COAT-FV) platelets. COAT-FV platelets are also observed on activation with thrombin plus collagen types I, V, or VI, but not with type III. No single agonist examined was able to produce COAT-FV platelets, although ionophore A23187 in conjunction with either thrombin or convulxin did generate this population. COAT-FV platelets bound annexin-V, indicating exposure of aminophospholipids and were enriched in young platelets as identified by the binding of thiazole orange. The functional significance of COAT-FV platelets was investigated by demonstrating that factor Xa preferentially bound to COAT-FV platelets, that COAT-FV platelets had more FV activity than either thrombin or A23187–activated platelets, and that COAT-FV platelets were capable of generating more prothrombinase activity than any other physiologic agonist examined. Microparticle production by dual stimulation with thrombin and convulxin was less than that observed with A23187, indicating that microparticles were not responsible for all the activities observed. These data demonstrate a new procoagulant component produced from dual stimulation of platelets with thrombin and collagen. COAT-FV platelets may explain the unique role of -granule FV and the hemostatic effectiveness of young platelets.

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.

scholarly journals Inhibition of platelet prothrombinase activity by a lupus anticoagulant

Blood ◽  
1983 ◽  
Vol 62 (1) ◽  
pp. 218-225 ◽  
Author(s):  
B Dahlback ◽  
IM Nilsson ◽  
B Frohm
Keyword(s):  
Blood Coagulation ◽  
Lupus Anticoagulant ◽  
Platelet Rich Plasma ◽  
Factor Xa ◽  
Inhibitory Effect ◽  
Factor Ix ◽  
Coagulation Cascade ◽  
Factor V ◽  
Coagulant Activity ◽  
Low Levels

Abstract Lupus anticoagulants are spontaneously occurring antibodies with specificity for negatively charged phospholipids. The plasma of a patient with such a polyclonal antibody of IgM type demonstrated low levels of factor VIII coagulant activity (VIII:C) and factors IX, XI and XII when analyzed by biologic clotting assays, whereas in immunochemical assays, normal levels of VIII coagulant antigen and factor IX were obtained. After immunoadsorption of patient plasma with anti-IgM Sepharose, normal biologic activities were demonstrated in clotting assays for VIII:C, factors IX, XI, and XII. The addition of the patient's isolated IgM to normal plasma resulted in grossly abnormal results in these coagulation assays, and a pattern similar to that of the patient's plasma was obtained. The inhibitory effect of the patient's lupus anticoagulant on blood coagulation was demonstrated also in platelet-rich plasma. The results of the clotting assays indicated that the anticoagulant inhibited several of the reactions in the blood coagulation cascade. The availability of purified components made it possible to demonstrate an inhibiting effect on the activation of prothrombin by factor Xa in the presence of isolated platelets, as well as in a system where purified factor V and well defined phospholipid vesicles were substituted for the platelets.

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