Factor X Activation by washed human platelets

1979 ◽  
Author(s):  
E van Wijk ◽  
L Kahlé ◽  
J ten Cate
Keyword(s):  
Human Factors ◽  
Trypsin Inhibitor ◽  
Calcium Ions ◽  
Factor Xa ◽  
Human Platelets ◽  
Soybean Trypsin Inhibitor ◽  
Thrombin Inhibitor ◽  
Chromogenic Substrate ◽  
Factor X ◽  
Factor X Activation

In a system of washed human platelets, Ca2+and purified human factors X anc II, a sufficient amount of thrombin is generated in about 10 minutes to aggregate the platelets. This thrombin is formed through the activation of FX by the platelets. In a system with either FX or FII present, no aggregation occurs. In addition no aggregation is observed when hirudin, a specific thrombin inhibitor, or when soybean trypsin inhibitor, which inhibits factor Xa, are added to the mixture. The formation of factor Xa can be monitored indirectly through the generation of thrombin, in the presence of an excess of prothrombin, using a thrombin sensitive chromogenic substrate. When washed platelets are incubated with FX alone for 10 minutes, no aggregation occurs and after the addition of prothrombin aggregation starts within 6 minutes. These findings confirm that washed platelets possess a factor X activating property. The generation of FXa proceeds in the absence of added Ca2+, whereas in the presence of Ca2+factor Xa activity reaches a maximum in 3 minutes, whereafter the activity progressively decreases. This may be due to the binding of Xa to the platelets in the presence of calcium ions.

Factor X Activation by Washed Human Platelets

1979 ◽  
Author(s):  
E.M. van Wijk ◽  
L.H. Kahlé ◽  
J.W. ten Cate
Keyword(s):  
Human Factors ◽  
Trypsin Inhibitor ◽  
Calcium Ions ◽  
Factor Xa ◽  
Human Platelets ◽  
Soybean Trypsin Inhibitor ◽  
Thrombin Inhibitor ◽  
Chromogenic Substrate ◽  
Factor X ◽  
Factor X Activation

In a system of washed human platelets, Ca2+ and purified human factors X an. II, a sufficient amount of thrombin is generated in about 10 minutes to aggregate the platelets. This thrombin is formed through the activation of FX by the platelets. In a system with either FX or FII present, no aggregation occurs. In addition no aggregation is observed when hirudin, a specific thrombin inhibitor, or when soybean trypsin inhibitor, which inhibits factor Xa, are added to the mixture. The formation of factor Xa can be monitored indirectly through the generation of thrombin, in the presence of an excess of prothrombin, using a thrombin sensitive chromogenic substrate. When washed platelets are incubated with FX along for 10 minutes, no aggregation occurs and after the addition of prothrombin aggregation starts within 6 minutes. These findings confirm that washed platelets possess a factor X activating property. The generation of FXa proceeds in the absence of added Ca2+, whereas in the presence of Ca2+ factor Xa activity reaches a maximum in 3 minutes, whereafter the activity progressively decreases. This may be due to the binding of Xa to the platelets in the presence of calcium ions.

scholarly journals Modulation of thrombin-mediated activation of factor VIII:C by calcium ions, phospholipid, and platelets

Blood ◽  
1985 ◽  
Vol 66 (1) ◽  
pp. 53-58
Author(s):  
MB Hultin
Keyword(s):  
Calcium Ions ◽  
Calcium Concentration ◽  
Human Platelets ◽  
Ionophore A23187 ◽  
Factor X ◽  
Platelet Activity ◽  
Platelet Surface ◽  
New Finding ◽  
Factor X Activation ◽  
External Calcium

The activation of factor VIII:C by thrombin appears to be an important prerequisite for the function of factor VIII:C as a cofactor in factor X activation in coagulation. The possible modulation of factor VIII:C activation by potential cofactors such as calcium ions, phospholipid, and platelets was studied systematically. Factor VIII:C activation could not be studied in the complete absence of Ca2+, since factor VIII:C activity decayed rapidly in calcium-free buffers, EDTA, or ethylene glycol tetra-acetic acid (EGTA), with only partial or no recovery of activity after readdition of Ca2+, Mn2+, or Mg2+. Added calcium chloride at 1.25, 2.5, 4, 10, 50, and 200 mmol/L produced progressive inhibition of factor VIII:C activation, with complete inhibition achieved by 50 mmol/L. Crude phospholipid preparations gave varying results, while purified phospholipids either had no effect or inhibited activation. This paper reports the new finding that fresh washed human platelets markedly potentiated factor VIII:C activation by a low concentration of thrombin (0.02 U/mL), even with prostaglandin E1 (PGE1) or dibutyryl cyclic AMP (cAMP) added to the washed platelets. However, the activity of platelets in factor VIII:C activation was inhibited by inclusion of PGE1 or dibutyryl cAMP during platelet washing, and ionophore A23187 increased this platelet activity; these data suggest that platelet stimulation is involved in the development of this activity. When platelets were maximally stimulated by thrombin (0.5 U/mL), the external calcium concentration increased 55 to 160 mumol/L, as measured with murexide, supporting the possible modulation of factor VIII:C activation by a transient increase in Ca2+ at the platelet surface.

scholarly journals Modulation of thrombin-mediated activation of factor VIII:C by calcium ions, phospholipid, and platelets

Blood ◽  
1985 ◽  
Vol 66 (1) ◽  
pp. 53-58 ◽  
Author(s):  
MB Hultin
Keyword(s):  
Calcium Ions ◽  
Calcium Concentration ◽  
Human Platelets ◽  
Ionophore A23187 ◽  
Factor X ◽  
Platelet Activity ◽  
Platelet Surface ◽  
New Finding ◽  
Factor X Activation ◽  
External Calcium

Abstract The activation of factor VIII:C by thrombin appears to be an important prerequisite for the function of factor VIII:C as a cofactor in factor X activation in coagulation. The possible modulation of factor VIII:C activation by potential cofactors such as calcium ions, phospholipid, and platelets was studied systematically. Factor VIII:C activation could not be studied in the complete absence of Ca2+, since factor VIII:C activity decayed rapidly in calcium-free buffers, EDTA, or ethylene glycol tetra-acetic acid (EGTA), with only partial or no recovery of activity after readdition of Ca2+, Mn2+, or Mg2+. Added calcium chloride at 1.25, 2.5, 4, 10, 50, and 200 mmol/L produced progressive inhibition of factor VIII:C activation, with complete inhibition achieved by 50 mmol/L. Crude phospholipid preparations gave varying results, while purified phospholipids either had no effect or inhibited activation. This paper reports the new finding that fresh washed human platelets markedly potentiated factor VIII:C activation by a low concentration of thrombin (0.02 U/mL), even with prostaglandin E1 (PGE1) or dibutyryl cyclic AMP (cAMP) added to the washed platelets. However, the activity of platelets in factor VIII:C activation was inhibited by inclusion of PGE1 or dibutyryl cAMP during platelet washing, and ionophore A23187 increased this platelet activity; these data suggest that platelet stimulation is involved in the development of this activity. When platelets were maximally stimulated by thrombin (0.5 U/mL), the external calcium concentration increased 55 to 160 mumol/L, as measured with murexide, supporting the possible modulation of factor VIII:C activation by a transient increase in Ca2+ at the platelet surface.

scholarly journals A Method for Measuring Activated Factor VIII in Plasma

1991 ◽  
Vol 66 (04) ◽  
pp. 430-434
Author(s):  
H Kessels ◽  
S Béguin ◽  
R Wagenvoord ◽  
H C Hemker
Keyword(s):  
Factor Viii ◽  
Plasma Sample ◽  
Factor Xa ◽  
Chromogenic Substrate ◽  
Factor X ◽  
High Concentration ◽  
Factor Viiia ◽  
Substrate Conversion ◽  
Simultaneous Inhibition ◽  
Factor X Activation

SummaryA method is described which enables a quantitative measurement of the concentration of activated factor VIII (VIIIa) in plasma. Based on the ability of factor VIIIa to accelerate the activation of factor X by factor IXa, phospholipid and calcium ions, the course of factor X activation in time is measured using a chromogenic substrate. Free factor Xa is able to activate non-activated factor VIII present in a plasma sample, which increases the factor X activation velocity, and thus disturbs the measurement of factor VIIIa. Furthermore, factor Xa was found to be inactivated by serine protease inhibitors from the plasma sample. By adding surplus chromogenic substrate these reactions of factor Xa are inhibited and at the same time the rate of substrate conversion is a measure of the amount of factor Xa present. Factor X activation and amidolysis of chromogenic substrate then take place simultaneously. It is shown that under proper conditions the factor X activation velocity is linearly proportional to the factor VIIIa concentration. This causes the optical density to increase as a parabolic function of time. The concentration of factor VIIIa can be obtained from the quadratic coefficient of the equation describing the parabola. The method is specific for factor VIIIa in that the extrinsic factor X activator is shown to have no influence on the measurement of factor VIIIa in thromboplastin activated plasma. We conclude that a sensitive and reliable method for assessing factor VIIIa concentrations in plasma has been developed on the basis of simultaneous inhibition and measurement of factor Xa by a high concentration of chromogenic substrate.

Heparin and Low Molecular Weight Heparins Inhibit Prothrombinase Formation but not its Activity in Plasma

1994 ◽  
Vol 72 (06) ◽  
pp. 862-868 ◽  
Author(s):  
Frederick A Ofosu ◽  
J C Lormeau ◽  
Sharon Craven ◽  
Lori Dewar ◽  
Noorildan Anvari
Keyword(s):  
Factor Xa ◽  
Catalytic Efficiency ◽  
Thrombin Inhibitor ◽  
Lag Phase ◽  
Factor V ◽  
Factor X ◽  
Normal Plasma ◽  
Plasma Factor ◽  
Prothrombin Activation ◽  
Plasma Heparin

SummaryFactor V activation is a critical step preceding prothrombinase formation. This study determined the contributions of factor Xa and thrombin, which activate purified factor V with similar catalytic efficiency, to plasma factor V activation during coagulation. Prothrombin activation began without a lag phase after a suspension of coagulant phospholipids, CaCl2, and factor Xa was added to factor X-depleted plasma. Hirudin, a potent thrombin inhibitor, abrogated prothrombin activation initiated with 0.5 and 1.0 nM factor Xa, but not with 5 nM factor Xa. In contrast, hirudin did not abrogate prothrombin activation in plasmas pre-incubated with 0.5,1.0 or 5 nM α-thrombin for 10 s followed by the coagulant suspension containing 0.5 nM factor Xa. Thus, thrombin activates plasma factor V more efficiently than factor Xa. At concentrations which doubled the clotting time of contact-activated normal plasma, heparin and three low Mr heparins also abrogated prothrombin activation initiated with 0.5 nM factor Xa, but not with 5 nM factor Xa. If factor V in the factor X-depleted plasma was activated (by pre-incubation with 10 nM a-thrombin for 60 s) before adding 0.5,1.0, or 5 nM factor Xa, neither hirudin nor the heparins altered the rates of prothrombin activation. Thus, none of the five anticoagulants inactivates prothrombinase. When 5 or 10 pM relipidated r-human tissue factor and CaCl2 were added to normal plasma, heparin and the three low Mr heparins delayed the onset of prothrombin activation until the concentration of factor Xa generated exceeded 1 nM, and they subsequently inhibited prothrombin activation to the same extent. Thus, hirudin, heparin and low Mr heparins suppress prothrombin activation solely by inhibiting prothrombinase formation.

scholarly journals Coupled amidolytic assay for factor VII: its use with a clotting assay to determine the activity state of factor VII

Blood ◽  
1978 ◽  
Vol 52 (5) ◽  
pp. 978-988 ◽  
Author(s):  
U Seligsohn ◽  
B Osterud ◽  
SI Rapaport
Keyword(s):  
Healthy Subjects ◽  
Activity Ratio ◽  
Factor Xa ◽  
Factor Vii ◽  
Test Material ◽  
Chromogenic Substrate ◽  
Factor X ◽  
Transfusion Therapy ◽  
The Mean ◽  
Activity State

Abstract A coupled amidolytic assay for factor VII (VII) has been developed that when used with a clotting assay for VII enables detection of activated VII. In the assay, VII in a test material determines generation of factor Xa in a mixture of purified factor X, tissue factor, and calcium; factor Xa is measured with a chromogenic substrate. Factor VII activity in the coupled amidolytic assay (VIIam) correlated well with VII activity in a one-stage clotting assay (VIIc) in 57 healthy subjects, 5 patients with hereditary VII deficiency, and 11 patients with liver disease. Activation of plasma VII by kaolin, clotting, or cold strikingly increased VIIc but not VIIam levels. Thus the ratio VIIc/VIIam (VII activity ratio) is a measure of VII activation. In 27 warfarin-treated patients the mean VII activity ratio was significantly decreased, reflecting a greater decline in VIIc than in VIIam. This probably stems from partially carboxylated VII being able to act during the 3-min incubation of the amidolytic assay but unable to act rapidly enough to affect the clotting assay. Measurement of VIIc/VIIam should enable evaluation of the activity state of VII in thrombotic disorders and in components for transfusion therapy.

scholarly journals The anticoagulant mechanism of action of heparin in contact-activated plasma: inhibition of factor X activation

Blood ◽  
1985 ◽  
Vol 65 (5) ◽  
pp. 1226-1231 ◽  
Author(s):  
TB McNeely ◽  
MJ Griffith
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Coagulation Factors ◽  
Factor Ix ◽  
Factor X ◽  
Clotting Time ◽  
Intrinsic Pathway ◽  
Blood Coagulation Factors ◽  
Factor Ixa ◽  
Factor X Activation

Abstract The effects of heparin on the activation of blood coagulation factors IX and X in contact-activated plasma were determined in the present study. In the presence and absence of 0.5 U/mL heparin, the amounts of factor IX that were cleaved 30 minutes after the addition of calcium and phospholipid to plasma exposed to glass (ie, contact activated) were essentially identical. In the absence of heparin, however, the plasma clotting time was between three and four minutes, while in the presence of heparin, the clotting time was approximately 40 minutes. More factor IXa was inhibited by antithrombin III in the presence of heparin than in its absence, but factor IXa levels sufficient for factor X activation appeared to be present in the heparinized plasma. Neither an increase in factor Xa nor a decrease in factor X was detected, however, in heparinized plasma. We conclude that the step in the intrinsic pathway of coagulation that is inhibited in the presence of heparin is at the level of factor X activation.

scholarly journals Activated clotting factors in factor IX concentrates

Blood ◽  
1979 ◽  
Vol 54 (5) ◽  
pp. 1028-1038 ◽  
Author(s):  
MB Hultin
Keyword(s):  
Antithrombin Iii ◽  
Initial Rate ◽  
Factor Xa ◽  
Factor Ix ◽  
Clinical Settings ◽  
Factor X ◽  
Clotting Factors ◽  
Factor Ixa ◽  
Human Factor Ix ◽  
Factor X Activation

Abstract The precise quantitation of activated factors in human factor IX concentrates has been accomplished with the use of recently developed, specific assays for factors IXa, Xa, and thrombin. The assay for factor IXa, which measures the initial rate of 3H-factor-X activation, was shown to be specific for factor IXa in the concentrates. Activated factor IX concentrates contained 1.0–2.3 microgram/ml of factor IXa; whereas the assays of unactivated concentrates were negative (less than 0.2 microgram/ml). The assays of factor Xa and thrombin, which measure the initial rate of p-nitroaniline release from S-2222 and S-2238, respectively, showed similar small amounts of factor Xa (4–34 ng/ml) and thrombin (12–76 ng/ml) in the activated and unactivated concentrates. The nonactivated partial thromboplastin time of the concentrates correlated significantly with the factor IXa content, but not with factor Xa or thrombin. Antithrombin III antigen in 3 of 4 concentrates was several-fold higher than antithrombin III activity, suggesting the presence of antithrombin III complexed with activated factors. These results support the hypothesis that the degree of activation of factor IX concentrates is related primarily to the concentration of factor IXa, which may be responsible for the thrombogenicity of these concentrates in some clinical settings.

scholarly journals In vivo studies of the role of factor VII in hemostasis

Blood ◽  
1985 ◽  
Vol 65 (5) ◽  
pp. 1197-1200
Author(s):  
AR Giles ◽  
S Tinlin ◽  
L Brosseau ◽  
H Hoogendoorn
Keyword(s):  
Alternative Pathway ◽  
Factor Xa ◽  
In Vivo Studies ◽  
Factor Vii ◽  
High Dose ◽  
Factor X ◽  
Clotting Factors ◽  
Dose Warfarin ◽  
Factor X Activation

The effect of both congenital and acquired factor VII deficiency on the cuticle bleeding time (CBT) was evaluated in dogs. The CBT has been previously documented to be a sensitive indicator of factor VIII:C deficiency in hemophilic dogs. Serial CBT determinations were made on normal dogs treated with high-dose warfarin. At 48 hours post- treatment, the CBT was normal, although the factor VII level was less than 1%, whereas the levels of factors II, IX, and X were 44%, 25%, and 17%, respectively. At 120 hours the CBT became abnormal when all vitamin K-dependent clotting factors had dropped to less than 18%. Administration of a plasma concentrate of factors II, IX, and X corrected the CBT, despite the factor VII level remaining at less than 1%. Similar studies in a congenitally factor VII-deficient dog (factor VII less than 2%) confirmed that this deficiency state was not associated with an abnormality of the CBT. Administration of heparin to both normal and factor VII-deficient animals was associated with prolongation of the CBT, but the heparin dose required in the normal animals was substantially higher than in the factor VII-deficient animals. These data do not suggest that factor VII/VIIa has an exclusive role in generating factor Xa, either directly or indirectly, by way of factor IXa generation, in vivo. However, the increase in heparin sensitivity of the factor VII-deficient animals does suggest that factor VII/VIIa may, in some circumstances, present a significant alternative pathway of factor X activation, although the activation pathway involved cannot be determined from the studies performed.

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