THE CONTRIBUTION OF ENDOGENOUS UROKINASE (UK) AND TISSUE PLASMINOGEN ACTIVATOR (t-PA) TO SPONTANEOUS CLOT LYSIS IN PLASMA

1987 ◽  
Author(s):  
V Gurewich ◽  
F Emmons ◽  
R Pannell
Keyword(s):  
Bacterial Growth ◽  
Dextran Sulfate ◽  
Platelet Rich Plasma ◽  
Clot Lysis ◽  
Lag Phase ◽  
Contact Activation ◽  
Human Fibrinogen ◽  
Tissue Plasminogen ◽  
The Uk ◽  
Time Point

Spontaneous lysis of 125I-labeled clots was measured in order to study the relative contributions of pro-UK, t-PA and contact activation. Clots were made up from 0.3 ml plasma, platelet rich plasma (PRP) or 2% human fibrinogen (Kabi) and suspended in plasma (3 ml) prepared from blood freshly collected onto citrate. The clots were preincubated to inactivate any residual thrombin which could inactivate pro-UK. Antibiotics were added to suppress bacterial growth. Lysis was approximately linear and went to completion with plasma clots in 12-16 d. and with fibrinogen clots in 6-10 d. Spontaneous lysis was inhibited by aprotinin at >200 KlU/ml and by antibody to t-PA. When the latter was added at t 2-5 d., clot lysis was arrested at each time point suggesting that t-PA was predominantly responsible for the lysis and not just for its initiation. Addition of antibody to UK or immunodepletion of plasma had little effect on spontaneous lysis of plasma or of fibrinogen clots, but retarded lysis of PRP clots. Only after ≥20 ng/ml of pro-UK were added to plasma, was a dose-responsive acceleration of clot lysis observed. The addition of antibodies to t-PA inhibited clot lysis by the added pro-UK (20-80 ng/ml) and greatly prolonged the lag phase of clot lysis by higher concentrations of pro-UK (100-200 ng/ml). Contact activation by dextran sulfate (1-5 μM) had no effect on spontaneous clot lysis but potentiated the effect of > 20 ng/ml added pro-UK. It was concluded that 1) The UK content of resting plasma (3-5 ng/ml) contributes little to lysis of platelet-free clots. 2) The presence of physiological concentrations of t-PA is essential for lysis by endogenous UK and markedly potentiates fibrinolysis by added pro-UK (20-200 ng/ml). 3) For endogenous UK to contribute to fibrinolysis, some amplification of its effect by a cofactor other than t-PA seems to be required. The results indicate augmentation of endogenous pro-UK induced fibrinolysis by platelets and that contact activation stimulates clot lysis by added pro-UK.

Adherent Mononuclear Cells And Activated Protein C (APC)

1981 ◽  
Author(s):  
F B Taylor ◽  
C T Esmon ◽  
R C Carroll ◽  
M L Lockhart
Keyword(s):  
Whole Blood ◽  
Fibrinolytic Activity ◽  
Mononuclear Cells ◽  
Degradation Products ◽  
Platelet Rich Plasma ◽  
Clot Lysis ◽  
Lag Phase ◽  
Polymorphonuclear Cells ◽  
Adherent Cells ◽  
Dose Dependent

Addition of bovine APC to dilute whole blood or euglobulin assays of fibrinolytic activity produces little effect on rates of clot lysis. However, addition of APC to undiluted non anti coagulated or to 3.2% citrated whole blood followed by 125I-fibrinogen and thrombin produces clots which release fibrin degradation products and lyse within 24 hours. Ordinarily clotted non-anticoagulated whole blood does not lyse.Addition of increasing amounts of APC from 12-100ug/ml increases the rate of whole blood clot lysis at least 20 fold over a 3.2% citrate control. This response is dose dependent with no evidence of saturation. Addition of increasing amounts of APC (25-200ug/ml) to platelet poor plasma (PPP) increases the rate of clot lysis only twofold. This increase is also dose dependent but is saturated at 80-100ug/ml. Addition of APC (60ug/ml) to 3.2% citrated PPP plus adherent mononuclear cells (1,000-2,000/MM3) again increases the rate of clot lysis at least 20 fold as with the whole blood system. However, this increase in rate is delayed. Substitution of platelet rich plasma (PRP) for PPP eliminates the lag phase. Substitution of non-adherent cells (1,000 MM3), RBC (3 million/MM3) or polymorphonuclear cells produces no acceleration of PPP clot lysis above the PPP-APC control. When deoxyglucose (1.4 × 10-1M) and anti-mycin (2.9 × 10-5M) are added to the PPP plus adherentxells before APC, the cell dependent acceleration of clot lysis is abolished. Removal of plasminogen activator (PA) and plasminogen (P) from PPP by lysine agarose adsorbtion also inhibits the effect of APC and adherent cells whereas reconstitution restores activity to 60%.We conclude that adherent mononuclear cells are required for expression of optimal APC pro-fibrinolytic activity and that this may require both P and PA. This suggests another role for mononuclear cells (monocytes) found in inflamatory and thrombotic lesions.

scholarly journals Impact of Deficiency of Intrinsic Coagulation Factors XI and XII on Ex Vivo Thrombus Formation and Clot Lysis

TH Open ◽  
2019 ◽  
Vol 03 (03) ◽  
pp. e273-e285 ◽  
Author(s):  
José W. P. Govers-Riemslag ◽  
Joke Konings ◽  
Judith M. E. M. Cosemans ◽  
Johanna P. van Geffen ◽  
Bas de Laat ◽  
...  
Keyword(s):  
Whole Blood ◽  
Thrombin Generation ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Coagulation Factor ◽  
Clot Formation ◽  
Clot Lysis ◽  
Contact Activation ◽  
Substantial Impact

AbstractThe contributions of coagulation factor XI (FXI) and FXII to human clot formation is not fully known. Patients with deficiency in FXI have a variable mild bleeding risk, whereas FXII deficiency is not associated with bleeding. These phenotypes make FXII and FXI attractive target proteins in anticoagulant therapy. Here, we studied the mechanisms of fibrin clot formation, stability, and fibrinolytic degradation in patients with severe FXI or FXII deficiency. Thrombin generation was triggered in platelet-poor (PPP) and platelet-rich plasma (PRP) with the biological FXII trigger sulfatides. Intrinsic and extrinsic thrombus formation and degradation in whole blood were determined with rotational thromboelastometry (ROTEM). Clot formation under flow was assessed by perfusion of whole blood over collagen microspots with(out) tissue factor (TF). Thrombin generation and clot formation were delayed in FXII- and FXI-deficient patients triggered with sulfatides. In FXI-deficient plasma, this delay was more pronounced in PRP compared to PPP. In whole blood of FXII-deficient patients, clots were smaller but resistance to fibrinolysis was normal. In whole blood of FXI-deficient patients, clot formation was normal but the time to complete fibrinolysis was prolonged. In flow chamber experiments triggered with collagen/TF, platelet coverage was reduced in severe compared with moderate FXI deficiency, and fibrin formation was impaired. We conclude that quantitative defects in FXII and FXI have a substantial impact on contact activation-triggered coagulation. Furthermore, FXI deficiency has a dose-dependent suppressing effect on flow-mediated and platelet/TF-dependent clot formation. These last data highlight the contribution of particularly FXI to hemostasis.

In Vitro Comparison of Fibrinolytic Activity of Plasminogen Activators Using a Thrombelastographic Method: In Vivo Evaluation of the B-Chain-Streptokinase Complex in the Dog Model Using Pre-Titered Doses

1986 ◽  
Vol 56 (01) ◽  
pp. 071-079 ◽  
Author(s):  
L Summaria ◽  
J Sandesara ◽  
G Yang ◽  
J P Vagher ◽  
J A Caprini
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Plasminogen Activators ◽  
Clot Lysis ◽  
In Vivo Evaluation ◽  
The Third ◽  
Dog Model ◽  
Tissue Plasminogen

SummaryThrombelastography was used to quantitatively compare the clot-lysing efficiency of 6 different plasminogen activators, using human whole blood, pooled normal plasma, and platelet rich plasma. The activators compared were the B-chain-streptokinase complex, the plasmin-streptokinase complex, the mini-plasmino-gen-streptokinase complex, tissue plasminogen activator, streptokinase, and urokinase. The most efficient activator found was the B-chain-streptokinase complex. This complex was 4.0 times more effective than streptokinase, 3.0 times more effective than the plasmin-streptokinase complex, 1.3 times more effective than the mini-plasminogen-streptokinase complex, 2.3 times more effective than tissue plasminogen activator, and 16.0 times more effective than urokinase. Although there were differences in both the coagulation and fibrinolysis thrombelastographic patterns between plasma and whole blood, the comparative efficiencies of each activator were the same with either plasma or bloodThe B-chain-streptokinase complex was evaluated as a thrombolytic agent in clot-lysis experiments in the jugular vein in the dog model, using a thrombelastographic method to determine the minimum dose of activator necessary for clot-lysis. With 6 dogs infused locally with 0.25 mg (8000 I.U.) of the plasmin-streptokinase complex, the cumulative clot-lysis was 18.0 ± 3.0% with the first dose, 33.0 ± 2.1% with the second dose, and 55.2 ± 8.6% with the third dose. With 6 dogs infused locally with 0.03 mg (2000 I.U.) of the B-chain-streptokinase complex, the cumulative clot-lysis was 30.6 ± 6.4% with the first dose, 54.4 ± 9.6% with the second dose, and 80.2 ± 9.0% with the third dose.

Platelet-Bound Prekallikrein Promotes Pro-Urokinase-Induced Clot Lysis: A Mechanism for Targeting the Factor XII Dependent Intrinsic Pathway of Fibrinolysis

1994 ◽  
Vol 71 (03) ◽  
pp. 347-352 ◽  
Author(s):  
Jean-Pierre Loza ◽  
Victor Gurewich ◽  
Michael Johnstone ◽  
Ralph Pannell
Keyword(s):  
Platelet Rich Plasma ◽  
Specific Inhibitor ◽  
Specific Effect ◽  
Clot Lysis ◽  
Factor Xii ◽  
Intrinsic Pathway ◽  
Clot Retraction ◽  
Enzymatic Mechanism ◽  
Low Concentrations ◽  
Factor Xiia

SummaryClots formed from platelet rich plasma were found to be lysed more readily by low concentrations of pro-urokinase (pro-UK) than clots formed from platelet poor plasma. This was not a non-specific effect since the reverse occurred with tissue plasminogen activator. A mechanical explanation due to platelet-mediated clot retraction was excluded by experiments in which retraction was inhibited with cyto-chalasin B. Therefore, a platelet-mediated enzymatic mechanism was postulated to explain the promotion of fibrinolysis. Casein autography of isolated platelets revealed a ≈ 90 kDa band of activity which comigrated with plasma prekallikrein (PK)/kallikrein, a known activator of pro-UK. Furthermore, treatment of platelets with plasma PK activator (PPA), consisting essentially of factor XIIa, induced activation of pro-UK and of chromomgenic substrate for kallikrein (S-2302). This activity corresponded to approximately 40-200 pM kallikrein per 10 8 washed and gel filtered platelets per ml. The activation of pro-UK by PPA-pretreated platelets was dose-dependent and inhibited by soybean trypsin inhibitor but not by bdellin, a specific inhibitor of plasmin, nor by the corn inhibitor of factor XIIa. Kinetic analysis of pro-UK activation by kallikrein showed promotion of the reaction by platelets. The KM of the reaction was reduced by platelets by ≈ 7-fold, while the kcat was essentially unchanged. In conclusion, PK was shown to be tightly associated with platelets where it can be activated by factor XIIa during clotting. The activation of pro-UK by platelet-bound kallikrein provides an explanation for the observed platelet mediated promotion of pro-UK-induced clot lysis. Since pro-UK and plasminogen have also been shown to be associated with platelets, the present findings suggest a mechanism by which the factor Xlla-dependent intrinsic pathway of fibrinolysis can be localized and targeted to a thrombus.

Conditions Affecting the Responses of Human Platelets to Epinephrine

1988 ◽  
Vol 60 (02) ◽  
pp. 209-216 ◽  
Author(s):  
Chantal Lalau Keraly ◽  
Raelene L Kinlough-Rathbone ◽  
Marian A Packham ◽  
Hidenori Suzuki ◽  
J Fraser Mustard
Keyword(s):  
Platelet Rich Plasma ◽  
Shape Change ◽  
Human Platelets ◽  
Dense Granule ◽  
Primary Phase ◽  
Lag Phase ◽  
Acid Citrate Dextrose ◽  
Two Phases ◽  
Citrate Dextrose ◽  
Thromboxane Formation

SummaryConditions affecting the responses of human platelets to epinephrine were examined. In platelet-rich plasma prepared from blood anticoagulated with hirudin or PPACK (D-pheny- lalanyl-L-prolyl-L-arginine chloromethyl ketone), epinephrine did not cause shape change or aggregation. In a Tyrode-albumin- apyrase solution containing a concentration of Ca2+ in the physiological range, and fibrinogen, epinephrine in concentrations as high as 40 μM did not induce platelet shape change, caused either no primary aggregation or very slight primary aggregation, and did not induce thromboxane formation, release of dense granule contents, or secondary aggregation. In contrast, in citrated platelet-rich plasma, epinephrine induced two phases of aggregation. This is not attributable to the generation of traces of thrombin since the same effects were evident when blood was taken into a combined citrate-hirudin anticoagulant or a combined citrate-PPACK anticoagulant. In a modified Tyrode-albu- min-apyrase solution containing approximately 20 μM Ca2+, 1 mM Mg2+, and fibrinogen, epinephrine induced extensive aggregation after a lag phase, but no primary phase was evident; thromboxane formation and release of dense granule contents accompanied the aggregation response. These responses were also observed when PPACK was included with the acid-citrate- dextrose anticoagulant, and in the washing and resuspending fluids. In the presence of aspirin or the thromboxane receptor blocker BM 13.177 a few small aggregates were detected by particle counting and by scanning electron microscopy; with the latter inhibitor, the platelets in the aggregates retained their disc shape; secondary aggregation and the responses associated with it did not occur. Thus thromboxane A2 formation is not necessary for the formation of these small aggregates, but is required for extensive aggregation and release. As with other weak agonists, the close platelet-to-platelet contact in the low Ca2+ medium appears to be necessary for full secondary aggregation. Omission of fibrinogen from the low Ca2+ medium prevented both primary and secondary aggregation in response to epinephrine. An antibody (10E5) to the glycoprotein Ilb/IIIa complex was completely inhibitory in the presence of fibrinogen. Thus the response of human platelets to epinephrine is influenced by the concentration of Ca2+ and the presence of fibrinogen in the medium in which they are suspended.

A Collaborative Study to Establish the 2nd International Standard for Tissue Plasminogen Activator (t-PA)

1987 ◽  
Vol 58 (04) ◽  
pp. 1085-1087 ◽  
Author(s):  
P J Gaffney ◽  
A D Curtis
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Collaborative Study ◽  
Chinese Hamster ◽  
World Health ◽  
Clot Lysis ◽  
Expert Committee ◽  
Single Chain ◽  
International Standard ◽  
Tissue Plasminogen

SummaryAn international collaborative study involving ten laboratories located in eight different countries was undertaken in order to replace the current International Standard (I.S.) for tissue plasminogen activator (t-PA). Two lyophilised candidate preparations of high purity were assessed in comparison with the current I.S. for t-PA using only a clot lysis assay. One preparation (coded 861670) was purified from a cultured melanoma cell supernatant and was about 98% single chain t-PA while the other preparation (coded 861624) was derived from Chinese hamster ovary (CHO) cells following DNA recombinant procedures and was 75% single chain t-PA.Both candidate preparations of t-PA compared in quite a satisfactory manner with the current I.S. from the viewpoint of the biometrics of parallel line bioassays and both preparations were quite stable for long periods at low temperatures and stable from up to 1 month at temperatures of 20° and 38° C. Both fultil the criteria to serve as a satisfactory Znd International Standard for t-PA. The Fibrinolysis Subcommittee of the International Committee for Thrombosis and Haemostasis recommended the melanoma source t-PA (861670) as the next I.S. in order to maintain continuity with the 1st I.S. which was also a melanomatype preparation. The data from the ten laboratories indicated that each ampoule of the new proposed standard contains 850 international units of t-PA activity by the clot lysis assay. It is planned to present the results of this study to the Expert Committee on Biological Standardization of the World Health Organization at its next meeting and to request that the preparation of t-PA, coded 861670, be established as the 2ndlnternational Standard for t-PA.

The Influence of Glycosylation on the Catalytic and Fibrinolytic Properties of Pro-Urokinase

1992 ◽  
Vol 68 (05) ◽  
pp. 539-544 ◽  
Author(s):  
Catherine Lenich ◽  
Ralph Pannell ◽  
Jack Henkin ◽  
Victor Gurewich
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cell ◽  
Human Gene ◽  
Culture Media ◽  
Mammalian Cell Culture ◽  
Glycosylation Site ◽  
Clot Lysis ◽  
Cell Culture Media ◽  
E Coli ◽  
The Uk

SummaryWe previously found that human pro-UK expressed in Escherichia coli is more active in fibrinolysis than recombinant human pro-UK obtained from mammalian cell culture media. To determine whether this difference is related to the lack of glycosylation of the E. coli product, we compared the activity of E. coli-derived pro-UK [(-)pro-UK] with that of a glycosylated pro-UK [(+)pro-UK] and of a mutant of pro-UK missing the glycosylation site at Asn-302 [(-) (302) pro-UK]. The latter two pro-UKs were obtained by expression of the human gene in a mammalian cell. The nonglycosylated pro-UKs were activated by plasmin more efficiently (≈2-fold) and were more active in clot lysis (1.5-fold) than the (+)pro-UK. Similarly, the nonglycosylated two-chain derivatives (UKs) were more active against plasminogen and were more rapidly inactivated by plasma inhibitors than the (+)UK.These findings indicate that glycosylation at Asn-302 influences the activity of pro-UK/UK and could be the major factor responsible for the enhanced activity of E. coli-derived pro-UK.

Functional Properties of p-Anisoylated Plasmin-Staphylokinase Complex

1993 ◽  
Vol 70 (02) ◽  
pp. 326-331 ◽  
Author(s):  
H R Lijnen ◽  
B Van Hoef ◽  
R A G Smith ◽  
D Collen
Keyword(s):  
Human Plasma ◽  
Rate Constant ◽  
Time Course ◽  
Bolus Injection ◽  
Similar Rate ◽  
Clot Lysis ◽  
Lag Phase ◽  
Plasma Clot ◽  
Rapid Clearance ◽  
Stoichiometric Complex

SummaryThe kinetic and fibrinolytic properties of a reversibly acylated stoichiometric complex between human plasmin and recombinant staphylokinase (plasmin-STAR complex) were evaluated. The acylation rate constant of plasmin-STAR by p-amidinophenyl-p’-anisate-HCI was 52 M-1 s-1 and its deacylation rate constant 1.2 × 10-4 s-1 (t½ of 95 min) which are respectively 50-fold and around 3-fold lower than for the plasmin-streptokinase complex. The acylated complex was stable as evidenced by binding to lysine-Sepharose. However, following an initial short lag phase, the acylated plasmin-STAR complex activated plasminogen at a similar rate as the unblocked complex, whereas the acylated plasmin-streptokinase complex did not activate plasminogen. These findings indicate that STAR, unlike streptokinase, dissociates from its acylated complex with plasmin in the presence of excess plasminogen. In agreement with this hypothesis, the time course of the lysis of a 125I-fibrin labeled plasma clot submerged in citrated human plasma, is similar for acylated plasmin-STAR, unblocked plasmin-STAR and free STAR (50% clot lysis in 2 h requires 12 nM of each agent). The plasma clearances of STAR-related antigen following bolus injection in hamsters were 1.0 to 1.5 ml/min for acylated plasmin-STAR, unblocked plasmin-STAR and free STAR, as a result of short initial half-lives of 2.0 to 2.5 min.The dissociation of the anisoylated plasmin-STAR complex and its consequent rapid clearance suggest that it has no apparent advantages as compared to free STAR for clinical thrombolysis.

Inhibition of Coagulation and Fibrinolysis by Aromatic Amidino Compounds

1971 ◽  
Vol 25 (03) ◽  
pp. 391-404 ◽  
Author(s):  
J.D Geratz
Keyword(s):  
Prothrombin Time ◽  
Human Plasma ◽  
Partial Thromboplastin Time ◽  
Inhibitory Effect ◽  
Clot Lysis ◽  
Similar Degree ◽  
Contact Activation ◽  
Plasma Clot ◽  
Human Thrombin ◽  
Canine Plasma

Summary1. Aromatic diamidines which are potent inhibitors of trypsin possess a marked inhibitory effect on the clotting activity of human thrombin and on the prothrombin time and partial thromboplastin time of human plasma. They also block the contact activation phase of the coagulation process. The strongest inhibitor among the compounds tested was M & B 4596 which was followed in second place by pentamidine.2. Pentamidine was 10 times more active than ε-ACA in impeding streptokinase-induced lysis of human plasma clots. It was 100-200 times stronger than ε-ACA in inhibiting the activation of bovine plasminogen by activators formed from the interaction between streptokinase and either human plasmin(ogen) or human plasma.3. The prothrombin time and partial thromboplastin time of canine plasma were less susceptible to inhibition by pentamidine than the same tests on human plasma. Clot lysis in the canine system was inhibited by pentamidine to a similar degree as in the human system. After intravenous injection of pentamidine in the dog there occurred the expected prolongation of the partial thromboplastin time and of the clot lysis time.

Tridegin, a Novel Peptidic Inhibitor of Factor XIIIa from the Leech, Haementeria ghilianii, Enhances Fibrinolysis In Vitro

1997 ◽  
Vol 77 (05) ◽  
pp. 0959-0963 ◽  
Author(s):  
Lisa Seale ◽  
Sarah Finney ◽  
Roy T Sawyer ◽  
Robert B Wallis
Keyword(s):  
Potent Inhibitor ◽  
Platelet Rich Plasma ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Fibrinolytic Enzymes ◽  
Small Polypeptide ◽  
Tissue Plasminogen ◽  
Protein Cross Linking

SummaryTridegin is a potent inhibitor of factor Xllla from the leech, Haementeria ghilianii, which inhibits protein cross-linking. It modifies plasmin-mediated fibrin degradation as shown by the absence of D-dimer and approximately halves the time for fibrinolysis. Plasma clots formed in the presence of Tridegin lyse more rapidly when either streptokinase, tissue plasminogen activator or hementin is added 2 h after clot formation. The effect of Tridegin is markedly increased if clots are formed from platelet-rich plasma. Platelet-rich plasma clots are lysed much more slowly by the fibrinolytic enzymes used and if Tridegin is present, the rate of lysis returns almost to that of platelet- free clots. These studies indicate the important role of platelets in conferring resistance to commonly used fibrinolytic enzymes and suggest that protein cross-linking is an important step in this effect. Moreover they indicate that Tridegin, a small polypeptide, may have potential as an adjunct to thrombolytic therapy.

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