The Nature of Synergy between Tissue-Type and Single Chain Urokinase-Type Plasminogen Activators

1989 ◽  
Vol 62 (03) ◽  
pp. 909-916 ◽  
Author(s):  
Edward T A Fry ◽  
David L Mack ◽  
Burton E Sobel
Keyword(s):  
Complex Formation ◽  
Plasma Proteins ◽  
Plasminogen Activators ◽  
Tissue Type ◽  
Single Chain ◽  
Urokinase Plasminogen Activators ◽  
Synergistic Combinations

SummaryEnhancement of thrombolysis with combinations of tissue-type and single chain urokinase plasminogen activators (t-PA and scu-PA) has been demonstrated in vivo but has not been seen consistently in vitro. This study was designed to characterize interactions between t-PA and scu-PA with respect to rate of and extent of thrombolysis in vitro and to delineate mechanisms responsible. Combinations of t-PA and scu-PA at selected concentrations synergistically enhanced thrombolysis in vitro compared with thrombolysis induced by either activator alone. Enhanced thrombolysis did not occur at the expense of fibrin specificity since the extent of fibrinogenolysis and consumption of α2-antiplasmin were significantly less with synergistic combinations of t-PA and scu-PA compared with equi-effective concentrations of either activator alone. Attenuation of complex formation of t-PA and two chain u-PA (tcu-PA), formed from scu-PA, with plasma proteins did not appear to contribute to enhancement of thrombolysis as assessed by fibrin autography. Binding of 125I-t-PA to thrombi was increased by 27% at 1 hr and by 21% at 2 hr in the presence of scu-PA (p <0,001 for both). Conversion of scu-PA to tcu-PA was enhanced when thrombi were exposed to scu-PA in the presence of t-PA. Results of this study indicate that t-PA and scu-PA at selected concentrations enhance thrombolysis in vitro synergistically without compromising fibrin specificity. Enhanced binding of t-PA to thrombi in the presence of scu-PA and enhanced conversion of scu-PA to tcu-PA appear to contribute to synergy between t-PA and scu-PA for thrombolysis.

ON THE MECHANISM OF THE IN VIVO SYNERGISM BETWEEN TISSUE-TYPE PLASMINOGEN ACTIVATOR (t-PA) AND SINGLE CHAIN UROKINASE-TYPE PLASMINOGEN ACTIVATOR (scu-PA)

1987 ◽  
Author(s):  
J M Stassen ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Rabbit Model ◽  
Sequential Therapy ◽  
Tissue Type ◽  
Single Chain ◽  
Molar Ratios ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

t-PA and scu-PA, in molar ratios between 1:4 and 4:1 do not act synergically in vitro (Thromb. Haemost. 56,35,1986) but display marked synergism in a rabbit model (Circulation 74, 838, 1986) and in man (Am. Heart J. 112, 1083, 1986). To investigate the mechanism of in vivo synergism in the rabbit model (J. Clin. Invest. 71, 368, 1983), t-PA and scu-PA were infused 1) simultaneously over 4 hrs, 2) t-PA over 1 hr, then 15 min later scu-PA over 2 hrs and 3) scu-PA over 1 hr, then 15 min later t-PA over 2 hrs.Significant synergism on thrombolysis is observed when t-PA and scu-PA are infused simultaneously or when t-PA is followed by scu-PA but not when scu-PA is followed by t-PA. These results suggest that low dose t-PA induces some plasminogen activation, sufficient to partially degrade fibrin, exposing COOH-terminal lysines with high affinity for plasminogen (Eur. J. Biochem. 140, 513, 1984). scu-PA might then activate surface-bound Glu-pla-minogen more efficiently.Sequential therapy with t-PA (or any other agent which "predigests" the thrombus), followed by scu-PA might constitute an alternative to simultaneous infusion of synergistic thrombolytic agents.

EVIDENCE FOR SYNERGISM BETWEEN TISSUE-TYPE PLASMINOGEN ACTIVATOR AND SINGLE CHAIN UROKINASE ON IN VITRO PLASMA CLOT LYSIS

1987 ◽  
Author(s):  
R S Rappaport ◽  
M R Blume ◽  
R L Vogel ◽  
M H Levner ◽  
P P Hung
Keyword(s):  
Plasminogen Activator ◽  
Tissue Type ◽  
Clot Lysis ◽  
Single Chain ◽  
Plasma Clot ◽  
Tissue Type Plasminogen Activator ◽  
Molar Ratios ◽  
Type Plasminogen Activator

There is mounting evidence from animal models and the clinic that combination thrombolytic therapy with tissue-type plasminogen activator (tPA) and single chain urokinase (scuPA) is synergistic. Yet, efforts to demonstrate synergism between these two plasminogen activators in vitro have met with discordant results. Collen et al (Thromb. Haemostasis, 56:35, 1986) reported an absence of synergism between these two agents on clot lysis in an in vitro plasma milieu when they were evaluated at molar ratios of 1:4 (tPA:scuPA and vice versa). Gurewich and Pannell (Thromb. Res., 44:217, 1986), however, reported a synergistic effect on fibrin-specific clot lysis in vitro when the agents were combined in concentrations exceeding molar ratios of 1:4 (tPA:scuPA). Here, we present evidence that synergism between tPA and scuPA may be demonstrated in vitro provided that the molar ratio of tPA to scuPA exceeds 1:4 and that the concentration of clot bound or unbound tPA is minimized. In order to achieve this experimental condition, the standard in vitro plasma clot lysis assay was modified. Human plasma clots were incubated first for a short time in plasma containing varying amounts of tPA. After incubation, the clots were washed thoroughly and reimmersed in plasma alone or in plasma containing varying amounts of scuPA or tPA. Under these conditions, lysis proceeded at a greater rate and to a greater extent when tPA clots were immersed in plasma containing an appropriate amount of scuPA than when they were immersed in plasma alone or in plasma containing appropriate amounts of tPA. Lysis of untreated clots or clots exposed first to scuPA and then to plasma containing varying amounts of scuPA proceeded far less efficiently with a characteristic lag. The enhanced lysis produced by tPA and scuPA obeyed the classical definition of synergy: the same biological effect can be obtained with two drugs together at algebraic fractional combinations of less than 1 (Berenbaum, M.C., Clin. Exp. Immunol., 28:1-18, 1977). Thus, conditions that more closely mimic the in vivo situation resulting from a bolus injection of tPA followed by infusion with scuPA, may provide a system for duplication of in vivo synergism in. vi tro and investigation of the mechanism thereof.

SYNERGISM, MUTANTS AND HYBRIDS OF TISSUE-TYPE PLASMINOGEN ACTIVATO(t-PA) AND SINGLE CHAIN UROKINASE-TYPE PLASMINOGEN ACTIVATOR(scu-PA):POTENTIAL FORTHROMBOLYTIC THERAPY

1987 ◽  
Author(s):  
D Collen
Keyword(s):  
Amino Acids ◽  
Clinical Investigation ◽  
Rabbit Model ◽  
Terminal Region ◽  
Tissue Type ◽  
In Vitro Mutagenesis ◽  
Single Chain ◽  
Thrombolytic Agents

With the development and clinical investigation of the fibrin-specific thrombolytic agents t-PA and scu-PA, many questions relating to their optimal use remain to be answered. It is, however, becoming apparent that these agents, in addition to several advantages,suffer some shortcomings, e.g. their therapeutic dose is large and their fibrin-specificity is limited.Therefore,the quest for better thrombolytic agents remains open.We will report results of four main lines of research which we are pursuing to obtain better agents or regimens for fibrin-specific thrombolytic therapy.1. Synergism between t-PA and scu-PA. t—PA and scu—PA in molar ratios between 4:1 and 1:4 show no synergism for thrombolysis of a plasma clot immersed in plasma in vitro(Thromb. Haemost. 56, 35, 1986) but display significant in vivo synergismin a rabbit model (Circulation 74, 838, 19867and in man (Am. Heart J. 112, 1083,1986).Recently we have confirmed synergism for thrombolysis between t-PA and scu-PA in a coronary thrombosis model in the dog(Zuskind et al., unpubl.) and in the baboon (Collen et al., unpubl.). Sequential infusion of t-PA followed by scu-PA butnot of scu-PA followed by t-PA is syneristic(Collen et al., this meeting).2. Mutants of t-PA. In collaboration with Larssen et al.,deletion mutants of t-PA, obtained by in vitro mutagenesis are characterized with respect to pharmacokinetics and thrombolytic properties.Mutants lacking the finger—like domain and/or the growth factor domainand/or one or all of the glycosylation sites have a much slower clearance (Larssenet al., this meeting) but unaltered specific thrombolytic properties and fibrin-specificity (Collen et al., this meeting).3. Mutants of scu-PA. A truncated form of scu-PA, lacking the 143 NH2~terminal amino acids was shown to be pharmacologically and thrombolytically indistinguishable from intact scu—PA (Stump et al.).Mutants of scu—PA in which Lys 158 is replaced,whereby they can no longer be converted to urokinase, still haveintrinsic plasminogen activating properties and act synergistically with t-PA on thrombolysis in vivo (Nelles et al., this meeting).4. Hybrids of t-PA and scu-PA. In collaboration with Pierard et al. (this meeting) hybrids of NH2~terminal regions of t-PA and COOH-terminal regions of u-PA were constructed which, after translation in transient expression systems, showed apparent specific activities comparable to that of natural two-chain u-PA. One hybrid, composed of the finger domain of t-PA and the B-chain of u-PA, was scaled up, purified and characterized (Gheysen et al., this meeting). This hybrid had theenzymatic properties typical of single chain u-PA, but had not acquired the fibrinaffinity of t-PA.Based on the finding that the isolated A-chain of t-PA retains the intact fibrin-affinity of the native molecule (Holvoet et al.,Eur.J. Biochem. 158, 173, 1986) andthat a low Mr form of scu-PA retains the functional properties of the intact moleule (J. Biol. Chem. 261, 17120, 1986), we have constructed and expressed a hybrid consisting of the NH -terminal region of t-PA (amino acids 1 to 263) and the COOH-terminal region of scu-PA (amino acids 144to 411) (Lijnen et al., this meeting). This hybrid has both fibrin affinity of t-PA (although less pronounced) and the enzymatic properties of scu-PA. The activation of plasminogen by the hybrid is apparently stimulated by fibrin.We believe that continued research along these lines will yield thrombolytic agents or therapeutic schemes, which may be superior to t-PA and/or scu-PA in terms of specific thrombolytic activity and fibrin-specificity.

scholarly journals Platelet Protease Nexin-1, a Serpin That Strongly Influences Fibrinolysis and Thrombolysis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 818-818
Author(s):  
Yacine Boulaftali ◽  
Benoit Ho-Tin-Noe ◽  
Ana Pena ◽  
Stéphane Loyau ◽  
Laurence Venisse ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Plasminogen Activators ◽  
Tissue Type ◽  
Dorsal Skinfold Chamber ◽  
Protease Nexin ◽  
Deficient Mice ◽  
Pai 1

Abstract Abstract 818 Fibrinolysis, a physiological process leading to clot resorbtion, is strictly controlled by fibrin-localized plasminogen activators (tPA and uPA) and by inhibitors like plasminogen activator type-1 (PAI-1). The serpin PAI-1 is a plasmatic serine protease inhibitor, that is also stored in platelets α-granules. PAI-1 inhibits both the action of urokinase- and tissue-type plasminogen activators (uPA and tPA respectively), and is up to now considered as the principal inhibitor of fibrinolysis in vivo. Interestingly, platelets are also known to inhibit fibrinolysis by both PAI-1-dependent and PAI-1-independent mechanisms. The individual role of other serpins, specifically protease nexin-1 (PN-1) in the thrombolytic process has not been investigated so far. Indeed, we recently demonstrated that a significant amount of PN-1 is stored within the α-granules of platelets and plays an antithrombotic function in vivo. PN-1, also known as SERPINE2, deserves a special interest since it also significantly inhibits in vitro uPA, tPA and plasmin. In this study, we explored the effect of PN-1 on fibrinolysis in vitro and in vivo. We evidenced the antifibrinolytic activity of platelet PN-1 in vitro using a specific PN-1-blocking antibody and PN-1 deficient platelets and, in vivo in PN-1−/− mice. Our data directly indicate that platelet PN-1 inhibits both tPA and plasmin activities in fibrin zymography. Remarkably, whereas fibrin-bound tPA or plasmin activity is not affected by PAI-1, we showed that PN-1 inhibits both plasmin generation induced by tPA-bound to fibrin and fibrin-bound plasmin. Moreover, PN-1 blockade or PN-1 deficiency result in an increased lysis of fibrin clots generated from platelet-rich plasma indicating that PN-1 regulates endogenous tPA-mediated lysis. Rotational thromboelastometry (ROTEM®) analysis shows that platelet PN-1 significantly decreases the rate of fibrinolysis ex vivo. Futhermore, blockade or deficiency of PN-1 provides direct evidence for an acceleration of the lysis-front velocity in platelet-rich clots. To challenge the role of PN-1 on fibrinolysis in vivo, we have developed an original murine model of thrombolysis. Using a dorsal skinfold chamber, thrombus formation induced by ferric chloride injury of venules and subsequent thrombolysis were visualized by microscopy on alive animals. This new approach allows a reproducible quantification of thrombus formation and of tPA- induced thrombus lysis. We observed that thrombi are more readily lysed in PN-1-deficient mice than in wild-type mice. Moreover, in PN-1 deficient mice, the rate and the extent of reperfusion were both increased (Figure A and B). These data demonstrate that platelet PN-1 is a new negative regulator of thrombolysis activity of plasmin, both in solution and within the clot. For the first time, this study shows that PN-1 protects towards thrombolysis and therefore could give rise to new approaches for therapeutic application. Indeed, PN-1 might be a promising target for optimizing thrombolytic therapy by tPA. Figure : Effect of PN-1 on thrombolysis. (A) Representative intravital images of vessels reperfusion after tPA treatment in dorsal skinfold chamber. (B) Quantification of the incidence of reperfused vessels within 1 hour post tPA treatment Figure :. Effect of PN-1 on thrombolysis. (A) Representative intravital images of vessels reperfusion after tPA treatment in dorsal skinfold chamber. (B) Quantification of the incidence of reperfused vessels within 1 hour post tPA treatment Disclosures: No relevant conflicts of interest to declare.

scholarly journals Thyroid follicular cells secrete plasminogen activators and can form angiostatin from plasminogen

2002 ◽  
Vol 173 (3) ◽  
pp. 475-481 ◽  
Author(s):  
JD Ramsden ◽  
S Yarram ◽  
E Mathews ◽  
JC Watkinson ◽  
MC Eggo
Keyword(s):  
Conditioned Medium ◽  
Angiogenesis Inhibitors ◽  
Plasminogen Activators ◽  
Tissue Type ◽  
Matrix Metalloproteinase 2 ◽  
Human Thyroid ◽  
Angiogenic Growth Factors ◽  
Thyroid Cells

Angiostatin, a 38 kDa fragment of plasminogen, potently inhibits the growth of blood vessels. Angiostatin is generated from plasminogen by urokinase-type (uPA) and tissue-type (tPA) plasminogen activators in the presence of free sulphydryl donors. Angiogenesis inhibitors may be important in regulating angiogenesis in developing goitre. We have examined angiostatin formation in human primary thyrocyte cultures and a rat thyrocyte cell line (FRTL-5). We found that human thyroid cells in culture secrete plasminogen activators (both tPA and uPA) as well as matrix metalloproteinase 2 into the medium. When human thyrocyte conditioned medium was incubated with plasminogen (10 microg/ml) and N-acetylcysteine (100 microM) for 24 h, a 38 kDa fragment of plasminogen, which is consistent with angiostatin, was generated. The appearance of the 38 kDa fragment was increased by agents that increase cAMP (forskolin and 8 BrcAMP). FRTL-5 cells, which do not secrete uPA or tPA, did not generate angiostatin. Thyroid cells produce several angiogenic growth factors, and human thyrocyte conditioned medium stimulated growth of endothelial cells. When the conditioned medium was incubated with plasminogen and N-acetylcysteine, this stimulatory effect was lost, consistent with the production of a growth inhibitory factor. We conclude that thyroid cells can produce angiostatin from plasminogen in vitro, and this may play a role in vivo in limiting goitre size.

Effect of Retraction on the Lysis of Human Clots with Fibrin Specific and Non-Fibrin Specific Plasminogen Activators

1989 ◽  
Vol 62 (04) ◽  
pp. 1083-1087 ◽  
Author(s):  
M Sabovic ◽  
H R Lijnen ◽  
D Keber ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Plasma Proteins ◽  
Plasminogen Activators ◽  
Tissue Type ◽  
Clot Lysis ◽  
Limiting Factor ◽  
Single Chain ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Blood Clots

SummaryThe effect of the serum content of human clots on their sensitivity to lysis with plasminogen activators was studied in a system composed of 125I-fibrin labeled clots immersed in buffer or in citrated plasma. The effect was studied with plasma clots before or after mechanical compression and with whole blood clots before or after retraction, using either the fibrin specific plasminogen activators recombinant tissue-type plasminogen activator (rt-PA) or recombinant single chain urokinase-type plasminogen activator (rscu-PA), and the non-fibrin specific activators recombinant two chain urokinase-type plasminogen activator (rtcu-PA), or streptokinase (SK).In a buffer milieu, all plasminogen activators had a similar fibrinolytic potency towards serum-rich clots (non-compressed plasma clots or non-retracted blood clots): 50% clot lysis in 4 h required 50 to 85 ng plasminogen activator per ml. Serum-poor clots (compressed plasma clots or retracted blood clots) were resistant to lysis in a buffer milieu but became sensitive to lysis following preincubation in plasma for 48 h. These findings indicate that plasma proteins entrapped in clots contribute significantly to their sensitivity to lysis and suggest that the amount of bound or entrapped plasminogen may be a limiting factor. In a plasma milieu, all plasminogen activators lysed serum- rich plasma or blood clots, albeit at higher concentrations (3 to 40 times higher than in the buffer milieu) and with different efficiencies: 50% clot lysis in 4 h required approximately 600 ng/ ml of rtcu-PA but 1,500 to 2,000 ng/ml of rscu-PA. These findings suggest that components of plasma are responsible for increased resistance of clots towards lysis and that the effect is variable for different plasminogen activators. Serum-poor plasma or blood clots were very resistant to lysis with non-fibrin specific agents, but became more sensitive after preincubation in plasma. However, serum-poor plasma or blood clots were sensitive to lysis with fibrin specific plasminogen activators, suggesting that during clot lysis with fibrin specific agents, plasminogen recruited from surrounding plasma may contribute significantly to clot lysis. The concentration of plasminogen activator required to obtain 50% clot lysis in a plasma milieu of compressed plasma clots or retracted blood clots was 390 and 1,600 ng/ml respectively for rt-PA and 1,100 and 3,200 ng/ml respectively for rscu-PA. These data suggest that in a plasma milieu retracted blood clots are more sensitive to lysis with fibrin specific plasminogen activators than with non-fibrin specific agents.

Fibrin-specificity of a Plasminogen Activator Affects the Efficiency of Fibrinolysis and Responsiveness to Ultrasound: Comparison of Nine Plasminogen Activators In Vitro

1999 ◽  
Vol 81 (04) ◽  
pp. 605-612 ◽  
Author(s):  
Dmitry V. Sakharov ◽  
Marrie Barrett-Bergshoeff ◽  
Rob T. Hekkenberg ◽  
Dingeman C. Rijken
Keyword(s):  
Thrombolytic Therapy ◽  
Plasminogen Activator ◽  
Tissue Type ◽  
Bolus Administration ◽  
High Frequency Ultrasound ◽  
Single Chain ◽  
Response Curves ◽  
Maximal Speed ◽  
Frequency Ultrasound

SummaryIn a number of cases, thrombolytic therapy fails to re-open occluded blood vessels, possibly due to the occurrence of thrombi resistant to lysis. We investigated in vitro how the lysis of hardly lysable model thrombi depends on the choice of the plasminogen activator (PA) and is accelerated by ultrasonic irradiation. Lysis of compacted crosslinked human plasma clots was measured after addition of nine different PAs to the surrounding plasma and the effect of 3 MHz ultrasound on the speed of lysis was assessed.Fibrin-specific PAs showed bell-shaped dose-response curves of varying width and height. PAs with improved fibrin-specificity (staphylokinase, the TNK variant of tissue-type PA [tPA], and the PA from the saliva of the Desmodus rotundus bat) induced rapid lysis in concentration ranges (80-, 260-, and 3,500-fold ranges, respectively) much wider than that for tPA (a 35-fold range). However, in terms of speed of lysis, these three PAs exceeded tPA only slightly. Reteplase and single-chain urokinase were comparable to tPA, whereas two-chain urokinase, anistreplase, and streptokinase were inferior to tPA. In the case of fibrin-specific PAs, ultrasonic treatment accelerated lysis about 1.5-fold. For streptokinase no acceleration was observed. The effect of ultrasound correlated with the presence of plasminogen in the outer plasma, suggesting that it was mediated by facilitating the transport of plasminogen to the surface of the clot.In conclusion, PAs with improved fibrin-specificity induce rapid lysis of plasminogen-poor compacted plasma clots in much wider concentration ranges than tPA. This offers a possibility of using single-or double-bolus administration regimens for such PAs. However, it is not likely that administration of these PAs will directly cause a dramatic increase in the rate of re-opening of the occluded arteries since they are only moderately superior to tPA in terms of maximal speed of lysis. Application of high-frequency ultrasound as an adjunct to thrombolytic therapy may increase the treatment efficiency, particularly in conjunction with fibrin-specific PAs.

Inhibition in Purified Systems and in Human Plasma of Chimaeric Plasminogen Activators Consisting of the NH2-Terminal Region of Tissue-Type Plasminogen Activator and the COOH-Terminal Region of UrokinaseType Plasminogen Activator

1988 ◽  
Vol 60 (02) ◽  
pp. 247-250 ◽  
Author(s):  
H R Lijnen ◽  
L Nelles ◽  
B Van Hoef ◽  
F De Cock ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Rate Constants ◽  
Plasminogen Activators ◽  
Second Order ◽  
Tissue Type ◽  
Single Chain ◽  
Chain Molecules ◽  
Order Rate ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

SummaryRecombinant chimaeric molecules between tissue-type plasminogen activator (t-PA) and single chain urokinase-type plasminogen activator (scu-PA) or two chain urokinase-type plasminogen activator (tcu-PA) have intact enzymatic properties of scu-PA or tcu-PA towards natural and synthetic substrates (Nelles et al., J Biol Chem 1987; 262: 10855-10862). In the present study, we have compared the reactivity with inhibitors of both the single chain and two chain variants of recombinant u-PA and two recombinant chimaeric molecules between t-PA and scu-PA (t-PA/u-PA-s: amino acids 1-263 of t-PA and 144-411 of u-PA; t-PA/u-PA-e: amino acids 1-274 of t-PA and 138-411 of u-PA). Incubation with human plasma in the absence of a fibrin clot for 3 h at 37° C at equipotent concentrations (50% clot lysis in 2 h), resulted in significant fibrinogen breakdown (to about 40% of the normal value) for all two chain molecules, but not for their single chain counterparts. Preincubation of the plasminogen activators with plasma for 3 h at 37° C, resulted in complete inhibition of the fibrinolytic potency of the two chain molecules but did not alter the potency of the single chain molecules. Inhibition of the two chain molecules occurred with a t½ of approximately 45 min. The two chain variants were inhibited by the synthetic urokinase inhibitor Glu-Gly-Arg-CH2CCl with apparent second-order rate constants of 8,000-10,000 M−1s−1, by purified α2-antiplasmin with second-order rate constants of about 300 M−1s−1, and by plasminogen activator inhibitor-1 (PAI-1) with second-order rate constants of approximately 2 × 107 M−1s−1.It is concluded that the reactivity of single chain and two chain forms of t-PA/u-PA chimaers with inhibitors is very similar to that of the single and two chain forms of intact u-PA.

Comparison of Specific Antibody, D-Phe-Pro-Arg-CH2Cl and Aprotinin for Prevention of In Vitro Effects of Recombinant Tissue-Type Plasminogen Activator on Haemostasis Parameters

1987 ◽  
Vol 58 (03) ◽  
pp. 921-926 ◽  
Author(s):  
E Seifried ◽  
P Tanswell
Keyword(s):  
Specific Antibody ◽  
Plasma Concentrations ◽  
Fibrinolytic Therapy ◽  
Tissue Type ◽  
Control Value ◽  
Type Plasminogen Activator ◽  
In Vitro Effects ◽  
Fibrinolytic Parameters

SummaryIn vitro, concentration-dependent effects of rt-PA on a range of coagulation and fibrinolytic assays in thawed plasma samples were investigated. In absence of a fibrinolytic inhibitor, 2 μg rt-PA/ml blood (3.4 μg/ml plasma) caused prolongation of clotting time assays and decreases of plasminogen (to 44% of the control value), fibrinogen (to 27%), α2-antiplasmin (to 5%), FV (to 67%), FVIII (to 41%) and FXIII (to 16%).Of three inhibitors tested, a specific polyclonal anti-rt-PA antibody prevented interferences in all fibrinolytic and most clotting assays. D-Phe-Pro-Arg-CH2Cl (PPACK) enabled correct assays of fibrinogen and fibrinolytic parameters but interfered with coagulometric assays dependent on endogenous thrombin generation. Aprotinin was suitable only for a restricted range of both assay types.Most in vitro effects were observed only with rt-PA plasma concentrations in excess of therapeutic values. Nevertheless it is concluded that for clinical application, collection of blood samples on either specific antibody or PPACK is essential for a correct assessment of in vivo effects of rt-PA on the haemostatic system in patients undergoing fibrinolytic therapy.

Purification of the Antihemophilic Factor by Gel Filtration on Agarose

1969 ◽  
Vol 22 (03) ◽  
pp. 577-583 ◽  
Author(s):  
M.M.P Paulssen ◽  
A.C.M.G.B Wouterlood ◽  
H.L.M.A Scheffers
Keyword(s):  
Factor Viii ◽  
Plasma Proteins ◽  
Large Scale ◽  
Gel Filtration ◽  
Large Scale Preparation ◽  
Scale Preparation ◽  
Antihemophilic Factor

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.

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