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Local delivery of antithrombotic drug prevents restenosis after balloon angioplasty in atherosclerotic rabbit artery.

We investigated the ability of various antithrombotic drugs, delivered locally, to prevent restenosis after angioplasty in hypercholesterolemic rabbits. After dilating atherosclerotic iliac stenoses by balloon angioplasty, a low dose of heparin or a new antithrombotic drug, such as low molecular weight heparin (fragmin), argatroban, or batroxobin, was delivered locally using the balloon double-occlusion technique. In 1 group, high-dose heparin was administered intravenously. Animals that received no drugs served as a control group. After angioplasty, the stenotic segment was dilated and the mean percentage luminal stenosis fell from 89% to 9% in the group that received locally delivered heparin, from 88% to 7% in the group that received locally delivered argatroban, from 87% to 11% in the group that received locally delivered fragmin, from 88% to 15% in the group that received locally delivered batroxobin, from 82% to 18% in the group that received i.v. heparin (p < 0.0001 compared with before angioplasty in each case), and from 84% to 17% in the control group (p < 0.005 compared with before angioplasty). Twenty-eight days after angioplasty, the percentage luminal stenosis remained at 14% in the group that received locally delivered argatroban, 15% in the group that received locally delivered fragmin, and 28% in the group that received locally delivered batroxobin, whereas it increased to 45% in the group that received i.v. heparin, 30% in the group that received locally delivered heparin and 72% in the control group (p < 0.05 compared with after angioplasty in each case). Thus, local delivery low doses of new antithrombotic drugs prevents restenosis after angioplasty without affecting systemic coagulability; heparin, whether administered locally or intravenously, was less effective than the new drugs in preventing restenosis.

Angiography↗

Effects of recombinant factor VIIa on platelet function and clot structure in blood with deficient prothrombin conversion.

While recombinant factor VIIa (rFVIIa) shows promise as a broad-spectrum hemostatic agent, questions remain regarding the most appropriate dose and the best way to monitor its effects. In this study we tested the sensitivity of a thrombin dependent platelet assay, platelet contractile force, to the effects of rFVIIa in normal, factor-deficient, and inhibitor-containing blood samples. Dose dependent effects of rFVIIa on platelet contractile force (PCF) and clot elastic modulus (CEM) were measured in all blood samples. rFVIIa minimally affected PCF and CEM in normal blood clotted with thrombin or batroxobin. While rFVIIa minimally altered PCF and CEM in factor VIII (FVIII) deficient blood clotted with thrombin, rFVIIa increased PCF and CEM and shortened the lag phase in a dose dependent manner in batroxobin-induced clots. The effects of rFVIIa in factor IX (FIX) deficient blood mirrored the effects seen in FVIII deficient samples. Whether clotted with thrombin or batroxobin, baseline PCF and CEM were abnormally low in FVIII deficient samples containing FVIII inhibitors. In such samples, rFVIIa caused dose dependent improvement of PCF, CEM, and lag phases. In one patient with a spontaneous inhibitor, rFVIIa caused dose dependent increases in PCF and CEM in blood clotted with either enzyme. rFVIIa corrects the deficient thrombin generation seen in FVIII and FIX deficiency, and in blood containing FVIII inhibitors. As a consequence, platelet function is improved and clot structure is enhanced. Platelet contractile force and clot elastic modulus measurements are sensitive to the dose dependent effects of rFVIIa.

Adult↗

[Modification of the toxicity of Bothrops atrox poison by interventions in the coagulation and kallikrein system of prey].

LD50-determinations with venom mixtures from different age groups of Bothrops atrox were carried out on differently pretreated mice. No differences were seen in the LD50 when comparing untreated mice with mice previously defibrinogenated with batroxobin. Inhibition of the coagulation-, kallikrein-kinin- and fibrinolytic system by pretreatment with batroxobin and aprotinin led to a significantly decreased toxicity of the venom mixtures of all age groups. The age-related difference in toxicity had practically disappeared. Furthermore, the number of mice of the latter group dying within one hour after the venom injection was strongly reduced as compared to untreated and only batroxobin-treated animals. Tests performed on rats showed that the rapidly occurring lethality following the venom injection as a consequence of circulatory disturbances (strong fall of arterial blood pressure, bradycardia, dyspnea), may be prevented almost completely by preincubation of the venom with aprotinin.

Age Factors↗

Anti-thrombotic effect of a PAI-1 inhibitor in rats given endotoxin.

The aim of this study was to investigate the anti-thrombotic effects of an inhibitor of the plasminogen activator inhibitor-1 (PAI-1) in rats given endotoxin. In studies in vitro, PRAP-1, a Fab-fragment of a polyclonal antibody against human PAI-1, was shown to inhibit PAI-1 activity in rat plasma as well as to stimulate clot-lysis of the euglobulin fraction derived from rat plasma. Endotoxin administered to anaesthetised rats produced a marked increase in plasma PAI-1 activity. To study fibrin formation and lysis in vivo after intravenous (i.v.) injection of the coagulant enzyme batroxobin, 125I-fibrinogen was administered to the animals. The thrombi formed by batroxobin were rapidly lysed in control animals, while the rate of lysis was markedly attenuated in rats given endotoxin. PRAP-1 was administered i.v. (bolus+infusion) to rats given endotoxin and batroxobin and the PAI-1 inhibitor caused a dose-dependent decrease in the 125I-fibrin deposition in the lungs. An immunohistochemical technique was used to confirm this decrease in density of fibrin clots in the tissue. Furthermore, PRAP-1 decreased plasma PAI-1 activity in the rats and this reduction was correlated to the decrease in lung 125I-fibrin deposition at the corresponding time point. It is concluded that in this experimental model the PAI-1 antibody PRAP-1 may indeed inhibit thrombosis in animals exposed to endotoxin.

Animals↗

Heparin ablates force development during platelet mediated clot retraction.

Although snake venom enzymes such as reptilase do not cause viscous metamorphosis, platelet secretion or clot retraction; when batroxobin and calcium are added to citrated blood significant platelet force development occurs. When this batroxobin-calcium system was applied to the study of platelet function during cardiopulmonary bypass (CPB), force development was found to be completely inhibited. After heparin reversal by protamine sulfate, significant recovery of force occurred. The present investigation was performed to evaluate the role of heparin in reducing force development during CPB. At concentrations above 0.10 U/ml, heparin totally suppressed force development in normal plasma. Addition of protamine sulfate to heparinized plasma caused complete recovery of force development. These concentrations of heparin had little effect on platelet aggregation by ADP or collagen. Possible direct effects of heparin on fibrin assembly and structure were studied by adding varying amounts of heparin to plasma and then inducing clot formation with batroxobin. At 1 U/ml, heparin reduced the size of fibrin fibers by 33%. Higher heparin concentrations had no additional effect. These results indicate that heparin may be responsible for a significant component of the decreased platelet force noted during cardiopulmonary bypass. To test whether heparin's effect could be due to suppression of thrombin activity, the effects of the antithrombin hirudin on force development were measured. Hirudin also inhibited force development in a concentration dependent manner. Thus, heparin's reduction of platelet force development may be due, at least in part, to suppression of thrombin activity.

Batroxobin↗

Prevention of postoperative pericardial adhesions with a defibrinogenating agent.

Pericardial adhesions pose a major problem during re-operative cardiac surgical procedures. The purpose of this study was to determine the effect of Batroxobin, a fibrinogen depleting agent, on the incidence of experimental pericardial adhesions. Twenty-four rabbits were divided into 3 groups of 8 rabbits each and pericardial mesothelial injury was induced by abrasion. Group A included rabbits receiving no further treatment, Group B included rabbits receiving intrapericardial injection of Ringer's solution, and Group C included rabbits receiving intrapericardial injection of Batroxobin. Three weeks after surgery, the incidence of adhesions in Groups B and C was compared with that in Group A. Pericardial adhesions found in 80% of the rabbits in Groups A and B. However, only 14% of the rabbits in Group C had pericardial adhesions (p < 0.05). Our findings demonstrated that intrapericardial Batroxobin reduced the incidence of pericardial adhesions in an animal model.

Animals↗

Trimeresurus stejnegeri snake venom plasminogen activator. Site-directed mutagenesis and molecular modeling.

The specific plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) is a serine proteinase presenting 23% sequence identity with the proteinase domain of tissue type plasminogen activator, and 63% with batroxobin, a fibrinogen clotting enzyme from Bothrops atrox venom that does not activate plasminogen. TSV-PA contains six disulfide bonds and has been successfully overexpressed in Escherichia coli (Zhang, Y., Wisner, A., Xiong, Y. L., and Bon, C. (1995) J. Biol. Chem. 270, 10246-10255). To identify the functional domains of TSV-PA, we focused on three short peptide fragments of TSV-PA showing important sequence differences with batroxobin and other venom serine proteinases. Molecular modeling shows that these sequences are located in surface loop regions, one of which is next to the catalytic site. When these sequences were replaced in TSV-PA by the equivalent batroxobin residues none generated either fibrinogen-clotting or direct fibrinogenolytic activity. Two of the replacements had little effect in general and are not critical to the specificity of TSV-PA for plasminogen. Nevertheless, the third replacement, produced by the conversion of the sequence DDE 96a-98 to NVI, significantly increased the Km for some tripeptide chromogenic substrates and resulted in undetectable plasminogen activation, indicating the key role that the sequence plays in substrate recognition by the enzyme.

Amino Acid Sequence↗

Effects of defibrination on hemorheology, cerebral blood flow velocity, and CO2 reactivity during hypocapnia in normal subjects.

BACKGROUND AND PURPOSE: Plasma fibrinogen is reported to be an independent risk factor for stroke and cardiovascular diseases. The effects of defibrination on hemorheology, middle cerebral artery (MCA) blood flow velocity, and CO2 reactivity during hypocapnia were evaluated in normal subjects. METHODS: Twenty-five healthy subjects (mean age, 31.8 +/- 5.7 years) were included in the study. Measurements were done at rest and repeated 24 hours after administration of 10 batroxobin units. Plasma fibrinogen, plasma viscosity, and whole blood viscosity were measured as hemorheological factors. MCA blood flow velocity was measured with a transcranial Doppler flowmeter. Blood flow velocity was corrected to 40 mm Hg of end-tidal CO2 partial pressure (PETCO2), and expressed as CV40. CO2 reactivity was measured as percent change in mean blood flow velocity per millimeter of mercury PETCO2. RESULTS: Plasma fibrinogen (from 7.04 to 2.29 mumol/L; P < .001), whole blood viscosity, and plasma viscosity decreased after administration of batroxobin. Mean MCA blood flow velocity at rest, CV40. and CO2 reactivity during hypocapnia increased significantly (from 67.4 to 73.6 cm/s, from 71.7 to 77.7 cm/s, and from 2.9%/mm Hg to 3.2%/mm Hg, respectively; P < .01) after defibrination. Mean arterial blood pressure and PETCO2 at rest were constant before and 24 hours after administration of batroxobin. There was a significant positive correlation between CV40 and CO2 reactivity (r = .623, P < .0001). CONCLUSIONS: The increase in MCA blood flow velocity was associated with improved CO2 reactivity and reduced blood viscosity after defibrination. The data may suggest that defibrination increases cerebral blood flow by reducing blood viscosity.

Adult↗

Dysfibrinogenemia and lupus anticoagulant in a patient with recurrent thrombosis.

We describe the coagulopathy of a 65-year-old woman with a thrombotic disorder associated with dysfibrinogenemia and lupus anticoagulant (LA). The patient's prothrombin time (PT), partial thromboplastin time (PTT), thrombin time (TT), and batroxobin time were prolonged and could not be corrected by mixing with equal volumes of normal plasma. Fibrinogen quantitation showed approximately twice as much immunoreactive as thrombin-clottable protein. The batroxobin and thrombin clotting times of the patient's isolated fibrinogen were prolonged and could not be corrected by mixture with normal fibrinogen. Turbidimetrically assessed fibrin monomer aggregation in response to thrombin, ancrod, or batroxobin and fibrin monomer reaggregation experiments disclosed clearly delayed onset and a lower maximum opacity. In other turbidimetric and clotting-time experiments, the patient's fibrinogen displayed a dose-dependent inhibition of the reaggregation of normal fibrin. Fibrinopeptide A and B release rates and sialic acid content were normal. Assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of reduced samples, the subunit structure of the patient's fibrinogen and its fully cross-linked fibrin was normal. The presence of LA was established by two techniques, the blood thromboplastin inhibition test and the platelet neutralization procedure (PNP). A positive PNP could not be produced by mixing afibrinogenemic plasma with the patient's purified fibrinogen. The patient's inactivated serum and her isolated IgG prolonged the PT and PTT of normal plasma but showed no inhibitory effect on the clotting of purified normal fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗

Local treatment with an antithrombotic drug reduces thrombus size in coronary and peripheral thrombosed arteries.

Since the treatment of thrombotic disease by antithrombotic drugs may be associated with bleeding complications, a local delivery technique for administration of the drug may be useful. The efficacy of low-dose local delivery of an antithrombotic drug on thrombosis was investigated in 73 dogs. The antithrombotic drug (heparin, 25 U/kg, antithrombin: argatroban, 0.05 mg/kg, or defibrinogenating agent: batroxobin, 0.05 U/kg) was infused locally to a 1-h-old thrombus, and no drug was given in controls. The effect of the local delivery on the thrombus was evaluated. Low- and high-dose systemic drug delivery was also evaluated. The mean reduction in thrombotic coronary stenosis observed by angiography was 30.3% with argatroban, 22% with heparin, and 20.8% with batroxobin (P < 0.005 vs controls). Systemic delivery of low-dose heparin or argatroban did not induce any change in thrombus size. With high-dose systemic drug delivery (heparin 250 U/kg, argatroban 0.5 mg/kg), the mean reduction of thrombotic stenosis was 15.2% with heparin and 32.8% with argatroban (P < 0.005 vs controls). In the iliac arterial thrombosis, after local delivery of the drugs, the mean reduction of thrombotic stenosis observed by angiography was 24.4% in the argatroban group, and 19.2% in the heparin group (P < 0.05 vs controls, respectively). With high-dose systemic heparin delivery, the mean reduction of the thrombotic stenosis was 13.2% (P < 0.01 vs control). Angioscopy also demonstrated a similar trend. The high-dose drug delivery reduced systemic coagulability. Thus, local delivery of an antithrombotic agent can reduce the thrombus size in the coronary and iliac arteries without having any significant influence on coagulability.

Angioscopy↗

Systemic haemorrhage in rats induced by a haemorrhagic fraction from Bothrops jararaca venom.

The main haemorrhagic fraction of Bothrops jararaca venom, showing in vitro fibrinogenolytic activity and an inhibitory effect on platelets aggregation induced by collagan, was studied in rats. Development of coagulopathy and/or haemorrhage was studied 2 hr after s.c. injection of batroxobin, B. jararaca whole venom and its haemorrhagic fraction. Incoagulable blood, together with low fibrinogen levels, were found only in rats injected with batroxobin and whole venom; thrombocytopenia alone was detected in rats given s.c. injections of haemorrhagin. Intravenous injection of low doses of haemorrhagin (less than 15 micrograms) resulted in significant thrombocytopenia, without any alterations in the blood coagulation mechanism. Severe damage to the vascular endothelium and skeletal muscle following s.c. injection of haemorrhagin together with signs of systemic haemorrhage in the kidneys, lungs and liver occurred. Levels of factor VIII and von Willebrand factor antigen were within the normal range in all animals. Serum levels of both whole venom and haemorrhagin were significantly correlated. This study confirms that B. jararaca haemorrhagin plays a vital role in systemic bleeding.

Animals↗

Fibrinogen and the early stages of polymerization to fibrin as studied by dynamic laser light scattering.

Human fibrinogen and the polymerization of fibrin after activation by the enzymes, thrombin and Batroxobin, were studied by means of dynamic laser light scattering (DLS). The apparent diffusion constant, D, for fibrinogen was measured and has a value of (1.80 +/- 0.42) X 10(-7) cm2 X s-1. D was found to contain contributions from the translational diffusion constant (Dt) as well as from the rotational diffusion constant (Dr). A comparison between experimental and calculated values of Dr and Dt suggests that fibrinogen in the absence of added Ca2+ expresses a certain degree of flexibility, while it is straightened in the presence of added Ca2+. The time dependence of D showed periodic oscillations, while the average D values decreased with time. Thrombin and Batroxobin caused similar behaviour of D. The period length was related to the enzyme concentration, clotting time (Ct) and the rate of release of fibrinopeptide A (FPA). No periodic oscillations were observed in experiments where the enzyme was replaced by saline, or in experiments using a dysfunctional fibrinogen (fibrinogen Aarhus) which displayed slow rates of FPA-release and polymerization. We propose that the periodic oscillations in a system far from equilibrium may be explained by conformational changes occurring in the fibrinogen molecule during enzyme activation and polymerization.

Batroxobin↗

Perioperative monitoring of primary and secondary hemostasis in coronary artery bypass grafting.

On-pump cardiac surgery is accompanied by complex alterations of hemostasis. The excessive postoperative bleeding has been attributed to acquired platelet dysfunction, impaired plasmatic coagulation, and increased fibrinolysis. The characterization of the hemostatic defects responsible for bleeding is crucial for specific treatment and optimal clinical management of the patient. For rapid determination of platelet-dependent primary hemostatic capacity (PHC), the Platelet Function Analyzer PFA-100 system is available. To evaluate the PFA performance in perioperative monitoring, a study was performed in 49 patients selected for low bleeding risk undergoing selective primary coronary artery bypass grafting (CABG). We compared PHC with Simplate bleeding time (BT) and platelet aggregometry. Furthermore, we analyzed global hemostasis by thromboelastography (TEG) and plasmatic coagulation by standard clotting tests prothrombin time (PT, Quick), activated partial thromboplastin time (aPTT), thrombin time (TT) and clotting factors and fibrinolysis by batroxobin (reptilase) time (RT). In all patients BT was postoperatively increased by 1.5- to 2-fold irrespective of perioperative complications and decreased to mildly prolonged values on the first postoperative day (1st day). In patients without complications, PHC in both collagen-adenosine diphosphate closure time (CADP-CT: 83 seconds preop, 78 seconds postop, and 74 seconds 1st day) and collagen-epinephrine closure time (CEPI-CT: 98 seconds preop, 95 seconds postop, 85 seconds 1st day) remained nearly stable. Apart from a patient with postoperative moderate thrombocytopenia, in bleeding patients no other significant defect of postoperative platelet hemostatic capacity was observed. However, on 1st day, the PHC of those patients was significantly reduced compared with non-bleeding patients. In patients with postoperative myocardial ischemia, increased PHC was identified by significantly shorter postoperative CADP-CT (66 seconds vs. 83 seconds) than in uncomplicated patients. By aggregometry, partial platelet dysfunction was observed in some patients without correlation to bleeding complications. In seven of 9 patients the postoperative bleeding complication was attributed to prolonged heparin anticoagulation and/or mildly enhanced fibrinogenolysis/fibrinolysis by TEG and standard plasmatic coagulation tests (TEG: k time 18 minutes vs. 8 minutes; aPTT: 47 seconds vs. 32 seconds; TT: 18.0 seconds vs. 12.3 seconds) and (RT: 19.5 seconds vs. 17.7 seconds). The impairment of PHC, platelet aggregation, and clotting factors observed on the 1st day in bleeding and in intra-aortic balloon pump (IABP) patients are most likely secondary effects, for example, loss of active platelets and clotting factors, to the primary postoperative bleeding or implantation of the IABP. In conclusion, our data indicate that in standard CABG procedures highly variable alterations of the hemostatic system occur after cardiopulmonary bypass (CPB) even in patients with assumed low operative risks. For identification of post-CPB bleeding complications, thromboelastography, aPTT, and TT and heparin and batroxobin (reptilase) time as fibrinolysis-sensitive assays are useful. Platelet function appears to be rapidly restored in uncomplicated CABG. PHC determination by PFA-100 demonstrates a high specificity for adequate platelet function and, therefore, could be beneficial in improved transfusion of platelet concentrates. PHC testing by PFA-100 may help identify postoperative platelet hyper-reactivity associated with myocardial lesion.

Adult↗

Hearing recovery in sudden deafness patients using a modified defibrinogenation therapy.

Ninety-eight patients with idiopathic sudden deafness were treated with a modified defibrinogenation (DF) therapy including batroxobin, low molecular dextran, vasodilators and vitamins. Hearing improvement was evaluated with two methods: categorical judgement and improvement rate (%). By categorical judgement, 60 patients (61% of the total) were classified into recovery or good improvement categories. The improvement rate was calculated for each of the 93 patients, and the average value was 64%. Modified DF therapy was effective especially for patients with severe hearing loss of 70-90 dB with flat audiogram. Although serum fibrinogen significantly decreased after batroxobin administration there was no correlation between the concentration of fibrinogen and hearing recovery. When prognostic factors were studied, the interval between the onset of hearing loss and start of treatment, initial hearing level, and the existence of vertigo all had significant correlation with the degree of hearing recovery.

Adenosine Triphosphate↗

[Preliminary study on the improvement of wound microcirculation and retrospection on several methods of the management of deep partial thickness burn wound].

OBJECTIVE: To analyze several methods of wound repair for deep partial thickness burn wounds retrospectively, so as to evaluate the significance of improvement of wound microcirculation on wound healing. METHODS: (1) 2,976 burn patients admitted to our department were enrolled in the study, among them 614 undertook tangential excision, 32, eschar abrasion, 86 allo-skin coverage after debridement, 1836 tropical application of silver sulfadiazine and 408 with traditional Chinese medicine (Jing Wan Hong ointment) with gauze bandage. The results of the management with different methods were compared. (2) Rat model with deep partial thickness burn was reproduced and topical application of silver sulfadiazine was given. The rats were randomly divided into control (n = 10, with normal saline injected via caudal vein within 5 minutes postburn), and treatment (n = 10, with batroxobin injected via caudal vein within 5 minutes postburn) groups. The blood flow perfusion unit in the wound skin was measured before burn and at 0.5 to 72 postburn hours by Laser Doppler. The wound healing rate, contraction rate and wound healing time in each group were calculated on 14 and 18 postburn days (PBDs). The number of hair follicles after wound healing was observed by histological method. RESULTS: (1) The burn wound treated by tangential excision healed within 2 to 3 post operation weeks (POWs), with the healing rate of 94.8% in patients with burn covering 50% - 70% TBSA and 93.4% in those with burn of 80% approximately 98% TBSA. The healing time of patients with allo-grafts coverage after eschar abrasion was 13.8 +/- 2.1 days without scar formation. The wound healing time was 18.0 +/- 2.3 day in 82 patients with allo-graft coverage after debridement, and it was 26.0 +/- 3.2 days with subeschar healing in 1658 patients with topical application of silver sulfadiazine. Infection in burn wound was encountered in most patients undergoing traditional Chinese medicine bandage treatment with wound healing time of 26.0 +/- 2.8 days in the lower extremities. (2) The blood flow perfusion unit of the rats in the treatment group was significantly higher than that in the control group (P < 0.01). The wound healing rate in treatment group on 14 and 18 PBD was obviously higher than that in the control group (P < 0.01). But the wound contraction rate in the two groups was similar (P > 0.05). The wound healing time in treatment group was much shorter than that in control group (P < 0.01). A few hair follicles remained in the dermis of the rats in the control group on 30 PBD, and the number was evidently smaller than that in the treatment group (P < 0.01). CONCLUSION: Early tangential excision and eschar abrasion remained better methods in the management of deep partial thickness burn wounds, as they could ameliorate burn wound infection, shorten treatment period, raise wound healing rate and quality. Application of batroxobin could accelerate wound healing rate by improving wound microcirculation in deep partial thickness burn wound.

Adult↗

Fibronectin and fibrin gel structure.

Plasma fibronectin is covalently incorporated into alpha-chains of fibrin gels in the presence of Factor XIII activated by thrombin (FXIIIaT) but not by Factor XIII activated by the snake venom enzyme batroxobin (FXIIIaB). FXIIIaB catalyzes introduction of gamma-gamma cross-links in fibrin but cross-linked alpha-chains are not formed. In the presence of FXIIIaT, fibrin gels formed by batroxobin incorporated fibronectin and the alpha-chains are cross-linked indicating that FXIIIaB has a different substrate specificity from FXIIIaT. In the presence of FXIIIaT the incorporation of fibronectin approaches 1 mol/340 kDa unit weight of fibrin. Fibronectin when present in a fibrinogen thrombin mixture containing FXIII does not influence the clotting time of the system nor the release of fibrinopeptides. Incorporation of fibronectin is not appreciable before the gel point. This indicates that the polymerization and gelation of fibrinogen is essentially not perturbed by the presence of fibronectin and that fibrin in the gel matrix rather than the fibrin polymers formed prior to gel point is the preferred structure for fibronectin incorporation. Incorporation of fibronectin into fibrin gels during formation leads to an increase in turbidity and a small decrease in Ks (permeability coefficient). This suggests that the width of the strands in the gel increases as a result of fibronectin incorporation. Fibronectin is also incorporated into preformed gels having completely cross-linked gamma- and alpha-chains perhaps indicating that the sites in fibrin involved in fibronectin incorporation are different from those involved in fibrin cross-linking. FXIIIaT appeared to be adsorbed to fibrin gel matrix in the presence but not in the absence of calcium ions.

Batroxobin↗

Platelets interact with fibrin only after activation.

Interactions between platelets and fibrin have been visualized by phase contrast, epifluorescence, and scanning electron microscope examination of clots formed with dansylcadaverine-labeled fibrin and gel-filtered platelets. After thrombin activation, the platelets appeared as fluorescent aggregates with bridging strands of fibrin; formaldehyde-fixed platelets were not fluorescent under the same experimental conditions. Scanning electron micrographs demonstrated that thrombin-activated cells had numerous pseudopods to which the fibrin strands adhered; fixed platelets exhibited a smooth discoid appearance and did not interact with the clot. Platelets trapped in clots formed with Batroxobin (Pentapharm) (platelets are not activated by Batroxobin as confirmed by light-scattering aggregometry measurements) remained as nonfluorescent, discoid cells, whereas platelets first activated by adenosine diphosphate formed brightly fluorescent aggregates. Light-scattering data of thrombin activation (0.2 U/mL) indicated that preincubation of platelets with 0.1 mmol/L prostaglandin E1 (PGE1) prior to addition of thrombin decreased the extent and rate of platelet shape change and resulted in 100-fold slower aggregation. Clots formed in the presence of PGE1 revealed decreased fluorescence intensity and fewer platelet-fibrin contacts. Gly-Pro-Arg-Pro, which blocks fibrinogen binding and fibrin assembly, was also effective in blocking platelet-fibrin interactions. These results indicate that platelet activation is a prerequisite for attachment of platelets to fibrin.

Adenosine Diphosphate↗

[Pharmacologic influence on secondary generalized fibrinolysis].

Secondary generalized hyperfibrinolysis was induced by thrombin infusion or batroxobin injection in rats. To follow intravascular fibrinolysis quantitatively, an electroimmuno-assay was used for determination of the fibrin degradation products formed. Anticoagulants (heparin, hirudin), antifibrinolytics (EACA, PAMBA, AMCA), and synthetic (APPA) and naturally occurring (aprotinin) protease inhibitors were studied with regard to their influence on secondary fibrinolysis. The potency and duration of action of the antifibrinolytics tested correspond to their antifibrinolytic activity measured in vitro and to their pharmacokinetics. Formation of degradation products is initiated after the appearance of fibrin monomer or fibrin, respectively. Due to their antithrombin action heparin, hirudin, and APPA prevent the thrombin-induced fibrin formation and thus the induction of secondary fibrinolysis. In contrast, formation of fibrin monomers caused by batroxobin is not influenced by thrombin inhibitors so that in this case formation of degradation products is not prevented.

Animals↗