Whole blood clot lysis: In vitro modulation by activated protein C

1985 ◽  
Vol 37 (6) ◽  
pp. 639-649 ◽  
Author(s):  
Fletcher B. Taylor ◽  
Marion S. Lockhart
Keyword(s):  
Whole Blood ◽  
Protein C ◽  
Blood Clot ◽  
Activated Protein C ◽  
Clot Lysis

scholarly journals The cofactor role of protein S in the acceleration of whole blood clot lysis by activated protein C in vitro

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 1189-1192 ◽  
Author(s):  
NJ de Fouw ◽  
F Haverkate ◽  
RM Bertina ◽  
J Koopman ◽  
A van Wijngaarden ◽  
...  
Keyword(s):  
Whole Blood ◽  
Protein C ◽  
Degradation Products ◽  
Blood Clot ◽  
Activated Protein C ◽  
Protein S ◽  
Tissue Type ◽  
Clot Lysis ◽  
Fibrin Degradation Products

Abstract The effect of purified human activated protein C (APC) and protein S on fibrinolysis was studied by using an in vitro blood clot lysis technique. Blood clots were formed from citrated blood (supplemented with 125I-fibrinogen) by adding thrombin and Ca2+-ions; lysis of the clots was achieved by adding tissue-type plasminogen activator. The release of labeled fibrin degradation products from the clots into the supernatant was followed in time. We clearly demonstrated that APC accelerates whole blood clot lysis in vitro. The effect of APC was completely quenched by antiprotein C IgG, pretreatment of APC with diisopropylfluorophosphate, and preincubation of the blood with antiprotein S IgG. This demonstrates that both the active site of APC and the presence of the cofactor, protein S, are essential for the expression of the profibrinolytic properties. At present, the substrate of APC involved in the regulation of fibrinolysis is not yet known. Analysis of the radiolabeled fibrin degradation products demonstrated that APC had no effect on the fibrin cross-linking capacity of factor XIII.

scholarly journals The cofactor role of protein S in the acceleration of whole blood clot lysis by activated protein C in vitro

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 1189-1192
Author(s):  
NJ de Fouw ◽  
F Haverkate ◽  
RM Bertina ◽  
J Koopman ◽  
A van Wijngaarden ◽  
...  
Keyword(s):  
Whole Blood ◽  
Protein C ◽  
Degradation Products ◽  
Blood Clot ◽  
Activated Protein C ◽  
Protein S ◽  
Tissue Type ◽  
Clot Lysis ◽  
Fibrin Degradation Products

The effect of purified human activated protein C (APC) and protein S on fibrinolysis was studied by using an in vitro blood clot lysis technique. Blood clots were formed from citrated blood (supplemented with 125I-fibrinogen) by adding thrombin and Ca2+-ions; lysis of the clots was achieved by adding tissue-type plasminogen activator. The release of labeled fibrin degradation products from the clots into the supernatant was followed in time. We clearly demonstrated that APC accelerates whole blood clot lysis in vitro. The effect of APC was completely quenched by antiprotein C IgG, pretreatment of APC with diisopropylfluorophosphate, and preincubation of the blood with antiprotein S IgG. This demonstrates that both the active site of APC and the presence of the cofactor, protein S, are essential for the expression of the profibrinolytic properties. At present, the substrate of APC involved in the regulation of fibrinolysis is not yet known. Analysis of the radiolabeled fibrin degradation products demonstrated that APC had no effect on the fibrin cross-linking capacity of factor XIII.

In vitro whole blood clot lysis by rt-PA is inversely correlated with baseline plasma levels of t-PA antigen

1994 ◽  
Vol 8 ◽  
pp. 43
Author(s):  
M. Colucci ◽  
S. Scopece ◽  
A. Gelato ◽  
L.G. Cavallo ◽  
N. Semeraro
Keyword(s):  
Whole Blood ◽  
Plasma Levels ◽  
Blood Clot ◽  
Clot Lysis

The Suppression of the Coagulation of Nonanticoagulated Whole Blood in vitro by Human Umbilical Endothelial Cells Cultivated on Microcarriers is not Dependent on Protein C Activation

1995 ◽  
Vol 73 (04) ◽  
pp. 719-724 ◽  
Author(s):  
Hans-Peter Kohler ◽  
Michele Müller ◽  
Thomas Bombeli ◽  
P Werner Straub ◽  
André Haeberli
Keyword(s):  
Endothelial Cells ◽  
Whole Blood ◽  
Protein C ◽  
Umbilical Vein ◽  
Activated Protein C ◽  
Volume Ratio ◽  
Endothelial Cell Surface ◽  
Direct Measurements ◽  
Umbilical Vein Endothelial Cells

SummaryHuman umbilical vein endothelial cells (HUVEC) were cultivated on globular microcarriers in order to improve the endothelial cell surface to blood-volume ratio over the conventional flat bed cultures. HUVEC-beads were tested for their modulation of blood coagulation using a combination of two steps: HUVEC-beads were added into the syringe used for the venipuncture, in order to achieve immediate contact between cells and blood, and no anticoagulant was used during the incubation time of HUVEC-beads with whole blood. The coagulation initiation produced by venipuncture was almost completely suppressed as judged by thrombin measurements over a period of 60 min. The activated partial thromboplastin time showed a prolongation by a factor >3. Direct measurements of activated protein C (APC) were negative. Moreover, inhibition of APC-generation with a monoclonal anti-human protein C antibody did not affect the anticoagulant properties of endothelial cells. Therefore the anticoagulant properties exerted by HUVEC-beads are not dependent on APC.

scholarly journals Determination of functional levels of protein C, an antithrombotic protein, using thrombin-thrombomodulin complex

Blood ◽  
1984 ◽  
Vol 63 (1) ◽  
pp. 15-21 ◽  
Author(s):  
PC Comp ◽  
RR Nixon ◽  
CT Esmon
Keyword(s):  
Protein C ◽  
Blood Clot ◽  
Activated Protein C ◽  
Mean Value ◽  
Clot Lysis ◽  
Functional Assay ◽  
Functional Protein ◽  
Normal Individuals ◽  
Patient Groups ◽  
Recurrent Venous Thrombosis

Abstract Protein C is a vitamin K-dependent plasma protein. Activated protein C is a potent anticoagulant and enhances blood clot lysis. We have developed a functional assay for protein C in human plasma. The measurement of protein C is accomplished by the addition of thrombomodulin, an endothelial-cell-associated cofactor for protein C activation, and thrombin in a 1:1 molar complex. The activated protein C formed in the plasma is immunoadsorbed with goat anti-human protein C IgG-agarose. The immunoadsorbed activated protein C retains the ability to hydrolyze chromogenic substrates, and after unbound plasma proteins are removed by washing, the bound activated protein C is quantitated by incubation with the substrate H-D-phe-pip-arg-p-nitroanilide (S-2238). Normal individuals have functional protein C levels of 3.9–5.9 micrograms/ml, with a mean value of 4.8 micrograms/ml. Individuals undergoing warfarin anticoagulation and patients with advanced liver diseases have decreased levels, as do certain patients with evidence of intravascular clotting. Functional protein C levels correlate well with immunologic levels of the protein in the patient groups studied. Heparin enhances the rate of activated protein C inhibition, as monitored by recovery of activated protein C by immunoadsorption. A patient with recurrent venous thrombosis and abnormal functional protein C activity, but normal levels of antigen, has been identified.

scholarly journals Alteration of fibrin network by activated protein C

Blood ◽  
1994 ◽  
Vol 83 (9) ◽  
pp. 2541-2548 ◽  
Author(s):  
A Gruber ◽  
E Mori ◽  
GJ del Zoppo ◽  
L Waxman ◽  
JH Griffin
Keyword(s):  
Protein C ◽  
Degradation Products ◽  
Activated Protein C ◽  
Relative Number ◽  
Relative Mass ◽  
Clot Lysis ◽  
Fibrin Degradation Products ◽  
Rate Of Increase ◽  
Fibrin Network

Abstract The antithrombotic plasma enzyme, activated protein C (APC), may play a role in thrombolysis. In vitro, acceleration of clot lysis by APC depends on its ability to inhibit the activation of prothrombin. The effect of APC on the assembly and dispersion of fibrin network was studied using turbidimetry, plasmin digestion of fibrin, and electron microscopy of plasma clots. The addition of APC before clotting but not after clotting accelerated clot lysis. The rate of increase in the turbidity of clotting plasma was reduced by APC. The turbidity of plasma clots containing APC was directly related to the clot lysis time. Fibrin from plasma clots that were formed in the presence of APC yielded less fibrin degradation products than fibrin from clots without added APC. Furthermore, APC reduced the diameter and relative number of fibrin fibers in plasma clots during gel assembly. We propose that APC may enhance the efficacy of thrombolysis by reducing the relative mass of fibrin within maturing thrombi.

scholarly journals Determination of functional levels of protein C, an antithrombotic protein, using thrombin-thrombomodulin complex

Blood ◽  
1984 ◽  
Vol 63 (1) ◽  
pp. 15-21
Author(s):  
PC Comp ◽  
RR Nixon ◽  
CT Esmon
Keyword(s):  
Protein C ◽  
Blood Clot ◽  
Activated Protein C ◽  
Mean Value ◽  
Clot Lysis ◽  
Functional Assay ◽  
Functional Protein ◽  
Normal Individuals ◽  
Patient Groups ◽  
Recurrent Venous Thrombosis

Protein C is a vitamin K-dependent plasma protein. Activated protein C is a potent anticoagulant and enhances blood clot lysis. We have developed a functional assay for protein C in human plasma. The measurement of protein C is accomplished by the addition of thrombomodulin, an endothelial-cell-associated cofactor for protein C activation, and thrombin in a 1:1 molar complex. The activated protein C formed in the plasma is immunoadsorbed with goat anti-human protein C IgG-agarose. The immunoadsorbed activated protein C retains the ability to hydrolyze chromogenic substrates, and after unbound plasma proteins are removed by washing, the bound activated protein C is quantitated by incubation with the substrate H-D-phe-pip-arg-p-nitroanilide (S-2238). Normal individuals have functional protein C levels of 3.9–5.9 micrograms/ml, with a mean value of 4.8 micrograms/ml. Individuals undergoing warfarin anticoagulation and patients with advanced liver diseases have decreased levels, as do certain patients with evidence of intravascular clotting. Functional protein C levels correlate well with immunologic levels of the protein in the patient groups studied. Heparin enhances the rate of activated protein C inhibition, as monitored by recovery of activated protein C by immunoadsorption. A patient with recurrent venous thrombosis and abnormal functional protein C activity, but normal levels of antigen, has been identified.

scholarly journals 3K3A-Activated Protein C Variant Does Not Interfere With the Plasma Clot Lysis Activity of Tenecteplase

Stroke ◽  
2020 ◽  
Vol 51 (7) ◽  
pp. 2236-2239
Author(s):  
Purba Mukherjee ◽  
Patrick Lyden ◽  
José A. Fernández ◽  
Thomas P. Davis ◽  
Kent E. Pryor ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Protein C ◽  
Activated Protein C ◽  
In Vitro Study ◽  
Tissue Type ◽  
Clot Lysis ◽  
Bolus Administration ◽  
Plasma Clot ◽  
Lysis Activity

Background and Purpose: A recombinant engineered variant of APC (activated protein C), 3K3A-APC, lacks anticoagulant properties (<10%) while preserving APCs anti-inflammatory, anti-apoptotic, and neuroprotective functions and is very promising in clinical trials for ischemic stroke. Therapeutic intervention with single bolus administration of the third-generation tPA (tissue-type plasminogen activator), tenecteplase, is anticipated to be widely adopted for treatment of acute ischemic stroke. 3K3A-APC is well-tolerated in stroke patients dosed with alteplase, and in vitro studies show 3K3A-APC does not interfere with alteplase-induced clot lysis. The purpose of this in vitro study was to assess the influence of 3K3A-APC on tenecteplase-induced clot lysis. Methods: Tenecteplase-mediated lysis of thrombin generated plasma clots of human normal pooled plasma was monitored in the presence of varying doses of 3K3A-APC. The effects on fibrinolysis by tenecteplase and alteplase were compared. Results: The presence of 3K3A-APC shortened the time for clot lysis induced by tenecteplase at very low levels but not at higher therapeutic concentrations of tenecteplase. Comparisons of alteplase-mediated clot lysis to tenecteplase clot lysis showed that both thrombolytic agents behaved similarly in the presence of 3K3A-APC. Conclusions: These results indicate that 3K3A-APC does not interfere with tenecteplase’s clot lysis function.

scholarly journals Inducing Acute Traumatic Coagulopathy In Vitro: The Effects of Activated Protein C on Healthy Human Whole Blood

PLoS ONE ◽  
2016 ◽  
Vol 11 (3) ◽  
pp. e0150930 ◽  
Author(s):  
Benjamin M. Howard ◽  
Lucy Z. Kornblith ◽  
Christopher K. Cheung ◽  
Matthew E. Kutcher ◽  
Byron Y. Miyazawa ◽  
...  
Keyword(s):  
Whole Blood ◽  
Protein C ◽  
Activated Protein C ◽  
Acute Traumatic Coagulopathy ◽  
Healthy Human ◽  
Human Whole Blood ◽  
Traumatic Coagulopathy

THE EFFECT OF PHYSICAL EXERCISE ON MONOCYTE FUNCTION, COAGULATION FACTORS, FIBRINOLYSIS AND PLATELETS

1987 ◽  
Author(s):  
B Ûsterud ◽  
J B Hansen ◽  
J O Olsen ◽  
L Wilsgård
Keyword(s):  
Blood Cell ◽  
Whole Blood ◽  
Cell Activation ◽  
Specific Activity ◽  
Blood Clot ◽  
Fibrinolytic System ◽  
Clot Lysis ◽  
Long Distance ◽  
National Team

Over a 2 years period, the Norwegian national team of cross country skiers, have been tested after strenous championships as well as before and after regular training.Finishing a 50 km race generated a rise of white cells from 5.4 ± 1.0 to 19.3 ± 3.7 × 109 /I (n=14). The mobilization of new and more sensitive white cells may explain the resultant rise in monocyte response to stimuli in vitro after the race. Thus, monocytes from blood incubated with 2 ng/ml blood, drawn from the athletes just after finishing the 50 km race, possessed 6-7 fold higher specific activity of thromboplastin than monocytes from blood drawn and stimulated in a rest-period. There was a positive correlation between the inverse level of F. VII in plasma of the skiers after the race and the monocyte response to stimuli in vitro as expressed by the level of thromboplastin. Activated monocytes with exposed thromboplastin are probably pulling out F. VII from the circulation just as seen in patients with gram negative septicaemia.A group of long distance runners were also tested after strenous jogging. High monocyte response to stimuli in vitro was associated with extremely active platelets that aggregated spontaneously after drawing the blood into heparin and tested it in a whole blood aggregometer. Those individuals with very active monocytes and platelets had also an extremely activation of their fibrinolytic system as judged by whole blood clot lysis. In contrast, everyone with low cell activation had hardly any change in their fibrinolytic activity after strenous running.The clear trend in this study was that almost everyone of our top athletes had a very depressed blood-cell activation system as compared to non athletes. Low blood cell activation in vivo was also associated with a low induction of the fibrinolytic system.

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