Effects of three cobra venoms on blood coagulation, platelet aggregation, and fibrinolysis

The effects of the venoms of Naja melanoleuca, Naja nigricollis, and Ophiophagus hannah on blood coagulation, platelet aggregation, and fibrinolysis were studied in vitro. All three venoms were shown to be anticoagulant. This action appeared to be due to an effect on both the extrinsic and blood thromboplastin mechanisms. Platelet aggregation in Chandler's tubes and adenosine diphosphate reactivity were inhibited by the three venoms, although in the case of Ophiophagus hannah venom they were inhibited only with intermediate concentrations. The three venoms possessed proteolytic properties, but when incorporated into purified caseinolytic systems and euglobulin clot lysis systems inhibition of plasmin activity was observed.     

Effects of the novel recombinant plasminogen activator BM 06.022 on human platelet aggregation in vitro

The present study was performed to evaluate effects of the novel recombinant plasminogen activator BM 06.022 on human platelet aggregation. BM 06.022 is an unglycosylated deletion variant of human tissue-type plasminogen activator. Aggregation was measured in platelet-rich plasma (PRP) from healthy human volunteers in response to adenosine diphosphate (ADP). Exposure of PRP to BM 06.022 induced a concentration-and time-dependent reduction of platelet aggregation compared with vehicle-control experiments. Incubation of high (3200 U/ml) concentrations of BM 06.022 for 0 to 15 min did not increase platelet aggregation. The platelet inhibitory effect of BM 06.022 could be reduced or abolished by aprotinin or chloromethylketone (PPACK), respectively, which is indicative of a plasmin-dependent mechanism. Concomitant incubation of BM 06.022, acetylsalicylic acid (ASA), and heparin decreased platelet aggregation less than BM 06.022 plus ASA alone, because heparin alone increased platelet aggregation. These findings demonstrate an inhibitory effect of BM 06.022 on platelet aggregation in citrated plasma, probably at least in part due to a plasmin-dependent mechanism.     

EDTA-dependent platelet phagocytosis. A cytochemical, ultrastructural, and functional characterization

Platelet satellitosis of polymorphonuclear cells is a phenomenon induced or enhanced by the anticoagulant EDTA. In contrast with previously reported studies, the subject in the present case did not demonstrate platelet satellitism but was profoundly pseudothrombocytopenic owing to platelet phagocytosis. Virtually all polymorphonuclear leukocytes and monocytes contained numerous ingested platelets in contrast with previous cases in which phagocytosis was observed only rarely and involved ingestion of single cells. The phenomenon was documented by immunocytochemical staining and transmission electron microscopy. Autoantibodies were detected in EDTA-anticoagulated blood. However, neither platelet antibody nor phagocytosis was present when heparin, acid-citrate dextrose, or citrate was used as an alternative anticoagulant. The antibody was not temperature dependent. Mixing studies showed the transfer of the phagocytosis phenomenon to healthy donors. Although platelet function assays are typically normal in EDTA-dependent platelet satellitism, this subject showed no secondary aggregation wave in response to adenosine diphosphate and depressed adenosine triphosphate release with collagen, adenosine diphosphate, and arachidonic acid.     

The influence of recombinant tissue-type plasminogen activator and plasmin on platelet aggregation: a quenched-flow study

Our study was designed to investigate the influence of human recombinant tissue-type plasminogen activator (rt-PA) and plasmin on platelet aggregation kinetics. Quenched-flow aggregometry coupled to single-particle counting was used with washed human platelets, which were pre-incubated for 5 min at 37°C with physiological and pharmacological doses of rt-PA (0.14–70 nM). Aggregation induced by adenosine diphosphate (ADP) under physiological flow conditions was followed over 15s. Depending on donor and reaction time, potentiation (one third of subjects), inhibition (one third of subjects), and no effect on ADP-induced (10 μM) aggregation were noted within the first 5s, while from 5.0 to 15.O s no influence was seen. On the other hand, using 2.5 μM ADP, aggregation was inhibited in all sensitive donors. Pre-incubation of platelets with plasmin (10nM, 100 nM) for 5 min at 37°C elicited a biphasic response to ADP. Depending on concentration and reaction time, plasmin potentiated or inhibited ADP-induced (10 μM) aggregation. Our data reveal that some individuals are susceptible for potentiation of platelet aggregation by both rt-PA and plasmin. Such susceptibility may contribute to the phenomenon of early reocclusion observed during thrombolytic therapy with rt-PA.     

Platelet aggregation: Part I Some effects of the adenosine phosphates, thrombin, and cocaine upon platelet adhesiveness

PLATELETS IN NATIVE BLOOD ADHERE SPONTANEOUSLY TO GLASS INDEPENDENTLY OF TEMPERATURE: if adenosine diphosphate is added to the blood the adhesiveness of the platelets is increased and this effect is largely independent of temperature. The mono- and triphosphates decrease adhesiveness at 20 degrees C. and 37 degrees C. but have no effect at 0 degrees C.; cocaine inhibits adhesion at 37 degrees C. and at 0 degrees C.Aggregation and viscous metamorphosis of platelets in native plasma is induced at 37 degrees C. by adenosine diphosphate or by thrombin; these reactions do not occur at 0 degrees C. Cocaine and all the other anti-adhesive drugs inhibit thrombin or adenosine diphosphate-induced aggregation. The mono- and tri-phosphates appear to compete with adenosine diphosphate and inhibit aggregation; they also inhibit thrombin-induced aggregation. Aggregation induced by adenosine diphosphate or thrombin is not prevented by any of the usual enzyme inhibitors or uncoupling agents at the appropriate strength. At 37 degrees C. aggregation and viscous metamorphosis induced by adenosine diphosphate or thrombin are reversible, and the addition of more adenosine diphosphate or of thrombin again produces aggregation and viscous metamorphosis.Platelets incubated with adenosine diphosphate but not agitated lose their power to aggregate but when more adenosine diphosphate is added with agitation, then aggregation is again produced. These observations are presumably explained by the finding that intact platelets, but not fragmented platelets, can inactivate adenosine diphosphate. From these results it is tentatively concluded that adhesion may involve intrinsic adenosine diphosphate in the platelet which may be activated by thrombin and inhibited by the added mono- or triphosphate. The anti-adhesive drugs act in a different manner. These phenomena have a remarkable similarity to those concerning mitochondrial swelling.     

Comparative Study of Modulatory Effects of Semax and Primary Proline-Containing Peptides on Hemostatic Reactions

Intranasal administration of Semax, peptide Pro-Gly-Pro, and a mixture of peptides Pro-Gly+Gly-Pro to rats for 5 days enhanced anticoagulant and fibrinolytic potential of the plasma (total fibrinolytic activity and plasmin and plasminogen activator activities) and decreased antiplasmin concentration. Semax and Pro-Gly-Pro decreased the weight of thrombi during experimental thrombosis.     

Fibrinogen degradation products generation is the major determinant of platelet inhibition induced by plasminogen activators in platelet-rich plasma

The platelet function defect induced by thrombolytic agents has been referred either to the degradation of platelet surface receptors or to the anti-aggregatory effect of fibrinogen degradation products (FgDPs).In the present study we have evaluated platelet aggregation induced by ADP, collagen and ristocetin after incubation of washed platelets or platelet-rich plasma (PRP) with plasmin (1.1–3.4IU/ml), plasminogen activators (PAs) (streptokinase 250–1000 IU/ml; urokinase, 10–1000 IU/ml; t-PA 0.5–10 μg/ml) or FgDPs (0.062–2 mg/ml). In parallel the surface levels of platelet GP lb and IIb/IIIa complex were determined by fluorescence flow cytometry using specific monoclonal antibody.Washed platelets treated with plasmin (1.1IU/ml) for 10 to 90 min showed a progressive reduction of ristocetin-induced platelet agglutination and a progressive reduction of surface GP Ib. Surface expression of GP IIb/IIIa complex was significantly increased after plasmin exposure.The addition of PAs to PRP resulted in a marked reduction of ADP-induced platelet aggregation. Collagen-induced platelet aggregation was only slightly affected. Similar changes were observed when PRP was preincubated with high concentrations of FgDPs. In PRP treated with PAs platelet surface levels of GP Ib and GP IIb/IIIa complex did not show any significant changes.In conclusion our results show that in plasma no proteolysis of platelet adhesive receptors occurs after plasminogen activation. The platelet inhibition observed after incubation of PRP with PAs is likely to be caused by FgDPs generation.     

Effect of fibrinogen degradation products on platelet aggregation

The digestion of fibrinogen with various concentrations of trypsin results in the formation of a variety of degradation products. Degradation products formed in this way have been purified by DEAE cellulose column chromatography and their effects on platelet aggregation investigated.TWO METHODS HAVE BEEN USED TO STUDY PLATELET AGGREGATION: a turbidimetric method which assesses platelet aggregation by the ability of adenosine diphosphate (ADP) to clump platelets and a method which assesses platelet adhesiveness by their ability to adhere to glass and to each other (modified Hellem technique, 1960).Three breakdown products produced by trypsin-digested fibrinogen were studied and all showed ;antithrombin' activity: two inhibited platelet aggregation, but one accelerated aggregation in both systems. Another product prepared by digestion of fibrinogen with urokinase-activated plasminogen has been shown to possess the ability to enhance ADP-induced platelet aggregation.     

中华眼镜蛇毒F组分抗血小板聚集的作用机制

目的探讨中华眼镜蛇毒F组分抑制血小板聚集的作用机制。方法用比浊法测定中华眼镜蛇毒F组分对二磷酸腺苷、花生四烯酸和血小板活化因子诱导血小板聚集作用的影响,流式细胞术观察中华眼镜蛇毒F组分对荧光标记的单克隆抗体CD41(FITC-CD41)和CD61(FITC-CD61)与血小板膜糖蛋白IIb/IIIa(GPIIb/IIIa)结合的影响。结果中华眼镜蛇毒F组分明显抑制二磷酸腺苷、花生四烯酸和血小板活化因子诱导的血小板聚集,其作用呈现一定程度的剂量依赖关系。中华眼镜蛇毒F组分可以明显降低单克隆抗体CD41(抗GPIIb)与血小板的结合率,而对单克隆抗体CD61(抗GPIIIa)与血小板的结合率没有影响。结论中华眼镜蛇毒F组分可以抑制多种激动剂诱导的血小板聚集,其机制和中华眼镜蛇毒F组分与血小板膜糖蛋白IIb/IIIa复合物的结合有关。     

中华眼镜蛇毒F组分抗血小板聚集的作用机制

目的 探讨中华眼镜蛇毒F组分抑制血小板聚集的作用机制.方法 用比浊法测定中华眼镜蛇毒F组分对二磷酸腺苷、花生四烯酸和血小板活化因子诱导血小板聚集作用的影响,流式细胞术观察中华眼镜蛇毒F组分对荧光标记的单克隆抗体CD41(FITC-CD41)和CD61(FITC-CD61)与血小板膜糖蛋白Ⅱb/Ⅲa(GPⅡb/Ⅲa)结合的影响.结果 中华眼镜蛇毒F组分明显抑制二磷酸腺苷、花生四烯酸和血小板活化因子诱导的血小板聚集,其作用呈现一定程度的剂量依赖关系.中华眼镜蛇毒F组分可以明显降低单克隆抗体CD41(抗GPⅡb)与血小板的结合率,而对单克隆抗体CD61(抗GPⅢa)与血小板的结合率没有影响.结论 中华眼镜蛇毒F组分可以抑制多种激动剂诱导的血小板聚集,其机制和中华眼镜蛇毒F组分与血小板膜糖蛋白Ⅱb/Ⅲa复合物的结合有关.     

Tissue plasminogen activator, plasminogen activator inhibitors, and activator-inhibitor complex in liver disease.

AIMS--To identify the relative contribution of plasminogen activators, particularly tissue plasminogen activator (t-PA) and specific plasminogen activator inhibitors (PAI-1, PAI-2), to the fibrinolytic changes associated with various types of liver disease or severe chemical and physical damage to the liver. METHODS--Platelet rich (PRP) and platelet poor plasma (PFP) from patients with alcoholic cirrhosis, primary biliary cirrhosis, hepatic malignancy, or paracetamol overdose, or who were undergoing partial hepatectomy or liver transplantation, were assayed for t-PA, PAI-1, t-PA-PAI-1 complex and PAI-2 antigen values using specific enzyme linked immunosorbent assays (ELISAs) developed in this laboratory. RESULTS--Appreciable increases in the plasma concentration of t-PA, PAI-1, and t-PA-PAI-1 were seen in patients with alcoholic cirrhosis, primary biliary cirrhosis, and hepatic malignancy. Liver damage due to paracetamol overdose and partial hepatectomy both resulted in a striking increase in plasma PAI-1 concentration, although concentrations of t-PA and t-PA-PAI-1 complex were less affected. Concentrations of t-PA, PAI-1, and t-PA-PAI-1 complex returned to near normal values after successful liver transplantation in a patient with chronic active hepatitis. PAI-2 was also detected in several patients with chronic liver disorders. CONCLUSIONS--Haemorrhage due to fibrinolytic bleeding is commonly associated with liver disease. The patients studied here all had appreciable increases in circulating t-PA antigen concentrations. This was associated with increased concentrations of PAI-1 antigen and t-PA-PAI-1 complex and the balance between activator and inhibitor did not result in systemic plasmin generation. Reduced PAI-1 activity in cirrhosis or a critical difference in the ratio of t-PA to PAI-1 concentrations may explain the enhanced plasminogen activator activity previously noted in cirrhosis but not metastatic disease. Reduced hepatic clearance of t-PA and t-PA-PAI-1 complex due to impaired liver function may account for increased concentrations of free and complexed t-PA.     

Factors responsible for the differential procoagulant effects of diverse plasminogen activators in plasma

This study was designed to define the procoagulant effects of different plasminogen activators in recalcified plasma. Accordingly, a two-stage assay procedure was developed in which citrated plasma was recalcified and incubated for 1–7 min with streptokinase (SK), urokinase (UK), or tissue-plasminogen activator (t-PA). An aliquot of this first-stage plasma was added to second-stage plasma; procoagulant activity was measured as the acceleration of the clotting time compared with that in control first-stage plasma to which plasminogen activators had not been added. With this procedure, second-stage clotting times were significantly accelerated after activation of plasminogen compared with those in control plasma and were shortest with 1000 i.u./ml SK. The presence of increased thrombin activity in the recalcified citrated plasma was verified by measurement of increased concentrations of fibrinopeptide A, which were inhibited by addition of I U/ml hirudin or or U/ml heparin to plasma during incubation with the plasminogen activators. Activation of factor X was induced in plasma incubated with SK. The rate of activation of factor X was increased when thrombin was elaborated. Induction of procoagulant activity was found to be dependent on depletion of α₂-antiplasmin and on the presence of plasmin activity in plasma, and could be inhibited by aprotinin. Thus, procoagulant activity in response to pharmacologic activation of plasminogen is dependent on plasmin-mediated activation of factor X, and subsequent activation of prothrombin.     

Comparison of textilinin-1 with aprotinin as serine protease inhibitors and as antifibrinolytic agents

Textilinin-1 (Q8008) was isolated from the venom of the Pseudonaja textilis and has a 47% sequence identity to the antihaemorrhagic therapeutic agent aprotinin. When equimolar concentrations of enzyme and aprotinin were pre-incubated, plasmin was inhibited 100%, plasma kallikrein 58%, and tissue kallikrein 99%. Under the same conditions, textilinin-1 inhibited plasmin 98%, plasma kallikrein 16% and tissue kallikrein 17%. Whole blood clot lysis was inhibited strongly by both aprotinin and textilinin-1, as shown by thrombelastography. At 2 microM inhibitor lysis initiated by t-PA was greater than 99% inhibited by aprotinin (LY60 = 0.4 +/- 0.1) whereas textilinin-1, inhibited lysis by 91% (LY60 = 8.9 +/- 0.7). The same trend was found with the lysis of euglobulin fractions. From these data textilinin-1 appears to be a more specific plasmin inhibitor than aprotinin but aprotinin inhibits clot lysis to a greater extent.     

Effects of peptide Pro-Gly-Pro-Leu under conditions of normal hemostasis and thrombus formation in rats

Tetrapeptide Pro-Gly-Pro-Leu in vitro effectively inhibited platelet aggregation over the entire range of studied concentrations (10⁻¹²–10⁻³ M). In concentrations of 10⁻⁹–10⁻³ M it exhibits fibrinolytic activity and in concentrations of 10⁻⁵–10⁻³ M has anticoagulant properties. Under in vivo conditions the analyzed tetrapeptide in a dose of 1 mg/kg increased anticoagulant, total and fibrin depolymerizating activities and increased activity of plasminogen activator. Intravenous administration produced more pronounced anticoagulant effect and leads to a greater increase in activity of plasminogen activator than intranasal administration. Tetrapeptide Pro-Gly-Pro-Leu also exerts antithrombotic effect. Preliminary repeated intranasal administration of the peptide before blood clot formation reduces the weight of fresh fibrin clots.     

Platelet release protein which inhibits plasminogen activators.

An inhibitor of plasminogen activator has been identified in human platelets by the technique of sodium dodecyl sulphate polyacrylamide gel electrophoresis and zymography. The inhibitor has a molecular weight of about 40 000 and is distinct from known plasma protease inhibitors. It is associated almost exclusively with platelets, with only trace amounts in platelet free plasma. The inhibitor is released during platelet aggregation or in vitro coagulation. This inhibitor inhibits both tissue type plasminogen activator and urokinase but has no effect on plasmin. It forms a 1:1 complex with tissue type plasminogen activator, which retains activity detectable under the analytical conditions used. A similar complex with urokinase either forms less readily or retains less activity.     

Identification of Two Laminin-Binding Fimbriae, the Type 1 Fimbria of Salmonella enterica Serovar Typhimurium and the G Fimbria of Escherichia coli, as Plasminogen Receptors

Escherichia coli strains carrying recombinant plasmids encoding either the type 1 fimbria of Salmonella enterica serovar Typhimurium or the G fimbria of E. coli exhibited binding of human ¹²⁵I-Glu-plasminogen and enhanced the tissue-type plasminogen activator-catalyzed conversion of plasminogen to plasmin. Purified type 1 or G fimbriae similarly bound plasminogen and enhanced its activation. The binding of plasminogen did not involve the characteristic carbohydrate-binding property of the fimbriae but was inhibited at low concentrations by the lysine analog -aminocaproic acid. Because these fimbrial types bind to laminin of basement membranes (M. Kukkonen et al., Mol. Microbiol. 7:229–237, 1993; S. Saarela et al., Infect. Immun. 64:2857–2860, 1996), the results demonstrate a structural unity in the creation and targeting of bacterium-bound proteolytic plasmin activity to basement membranes.     

Bacterial plasminogen receptors: in vitro evidence for a role in degradation of the mammalian extracellular matrix.

The potential of bacterium-bound plasmin to degrade mammalian extracellular matrix and to enhance bacterial penetration through basement membrane was assessed with the adherent strain SH401-1 of Salmonella enterica serovar Typhimurium. Typhimurium SH401-1 was able to bind plasminogen and to enhance the tissue-type plasminogen activator-mediated activation of the single-chain plasminogen to the two-chain plasmin. The end product, the enzymatically active, bacterium-bound plasmin activity, was also formed in a normal human plasma milieu in the presence of exogenous tissue-type plasminogen activator, indicating that plasmin was protected from the plasminogen activator inhibitors and plasmin inhibitors of plasma. Plasmin bound on Typhimurium cells degraded 125I-labeled laminin as well as 3H-labeled extracellular matrix prepared from the human endothelial cell line EA.hy926. The degradations were not seen with Typhimurium cells without plasminogen and were inhibited by the low-molecular-weight plasmin inhibitor aprotinin. Plasmin bound on Typhimurium cells also potentiated penetration of bacterial cells through the basement membrane preparation Matrigel reconstituted on membrane filters. The results give in vitro evidence for degradation of the mammalian extracellular matrix by bacterium-bound plasmin and for a pathogenetic role for bacterial plasminogen receptors.     

Interaction of a group A Streptococcus within human plasma results in assembly of a surface plasminogen activator that contributes to occupancy of surface plasmin-binding structures

Group A streptococcal isolate 187061 incubated in human plasma or serum reconstituted with fibrinogen but not plasminogen-depleted plasma or serum alone acquired a surface plasminogen activator activity. Assembly of the surface plasminogen activator was inhibited by the presence of neutralizing antibodies to streptokinase. Once assembled, the bacterial-associated plasminogen activator could generate plasmin when incubated in human plasminogen, plasmin or serum which could bind to bacterial surface plasmin-binding structures despite the presence of host physiological inhibitors. These studies provide evidence that the pathways by which group A isolates interact with human plasmin(ogen) are potentially linked and may provide a mechanism for bacteria to acquire host enzymatic activity efficiently in the infected host.     

Presence of direct thrombin inhibitors can affect the results and interpretation of lupus anticoagulant testing

In vitro experiments were conducted to determine whether the direct thrombin inhibitors argatroban and lepirudin can interfere with the results of lupus anticoagulant (LA) testing. Concentration-response curves were generated to calculate the concentration of anticoagulant that prolongs the activated partial thromboplastin time (aPTT) to 75 seconds (2.5 times the baseline average). Corresponding concentrations of anticoagulant were added to plasma samples before running dilute Russell viper venom tests (DRVVTs) and LA-sensitive aPTTs (PTT-LAs). Because the DRVVT test system contains an antiheparin agent, DRVVT results were not prolonged in the presence of heparin. With argatroban added to normal plasma samples, neither the DRVVT percent correction of ratio nor the DRVVT test/confirm ratio were elevated, but when added to LA-positive plasma, some false-negative results were observed. Lepirudin increased the DRVVT percent correction of ratio and the DRVVT test/confirm ratio into a range that could lead to false-positive identifications of LAs. In sharp contrast to the DRVVT test system, distinction between LA-positive and LA-negative plasma samples was maintained and possibly even enhanced in the platelet neutralization procedure correction phase of the PTT-LA test system.     

The activation of plasminogen by tissue plasminogen activator is not enhanced by different heparin species as assessed in vitro with a solid-phase fibrin method

The aim of this study was to evaluate the effect of several heparin species (standard heparin, heparin of low molecular weight: IC 831422, heparin of high affinity for antithrombin III: IC 831435, and heparin of low affinity for antithrombin III: IC 831436) on the different steps of the fibrinolytic mechanism i.e., interaction of tissue-type plasminogen activator (t-PA) with plasminogen activator inhibitor-1 (PAI-1), binding of t-PA and plasmin(ogen) to fibrin and activation of plasminogen on the fibrin surface, in the presence of plasma proteins and factors that modulate fibrinolysis. Fibrinolytic and plasmin amidolytic activities were measured in the presence and absence of heparin. The spectrophotometric assays were performed in the presence and absence of solid-phase fibrin using selective chromogenic substrates for plasmin and saturating concentrations of plasminogen.The overall fibrinolytic activity, the amidolytic activity of mixtures of t-PA or urokinase with plasminogen in the absence of fibrin, the binding of t-PA and plasmin(ogen) to fibrin and the t-PA/PAI-1 interaction were not modified by heparin. In contrast, the activation of plasminogen by fibrin-bound t-PA decreased as a function of the concentration of heparin. Although this effect was not significant at concentrations usually used in routine heparin therapy (0.1 to 1 iu/ml) it might have some relevance when heparin is injected in bolus doses.In conclusion, by using a solid-phase fibrin method which allows the analysis of t-PA/PAI-1 balance, data were obtained indicating that the heparin species tested here neither competed with fibrin for the binding of t-PA, nor potentiated the activation of plasminogen at the fibrin surface.