Lysing patterns of retracted blood clots with diffusion or bulk flow transport of plasma with urokinase into clots--a magnetic resonance imaging study in vitro.

Fresh retracted clots are known to be poorly lysable by fibrinolytic agents. We have studied whether lysis of retracted clots could be enhanced by bulk transport in comparison to pure diffusion of plasma containing urokinase (400 IU/ml) into the clots. Cylindrical retracted blood clots were occlusively glued by a polyester into plastic tubes and put in contact with plasma through the clot bases. One group of clots (perfused clots, n = 10) was placed under a pressure difference of 6 kPa (60 cm H2O) which resulted in an average plasma flow of 0.97 +/- 0.34 microliters/min through the clot during the first hour. Another group of clots (non-perfused clots, n = 10) was incubated in the lytic plasma without a pressure difference. Clot sizes were measured during lysis by magnetic resonance imaging (MRI). Channels representing lysed areas penetrated into perfused clots with a velocity of 5.4 +/- 1.6 mm/h (n = 10), whereas the boundaries of non-perfused clots subsided with a velocity of less than 0.1 mm/h. Eight of the 10 perfused clots were recanalized after 8 h and the sizes of the perfused group were reduced to 64.0 +/- 10.7% of the initial values. The relative sizes of non-perfused clots after 8 h remained significantly higher: 95.0 +/- 1.3%, p < 0.005. In a separate experiment good agreement was obtained between the measured clot sizes by MRI and the residual radioactivity of 125I-fibrin in the clot.(ABSTRACT TRUNCATED AT 250 WORDS)     

尿激酶溶凝时效性和量效性的体外研究

目的探讨不同剂量尿激酶溶解体外血凝块的效果,寻找最佳溶凝时间和用药剂量,为临床上合理应用尿激酶溶解液化颅内血肿提供实验依据。方法参照临床常见的高血压脑内血肿出血量,取180例健康志愿者静脉血60m1,加尿激酶0．5、1、2、5、10及15万U,测量加药后1、2、3、4、5、6h的溶凝效果,分析尿激酶最佳溶凝时间和用药剂量。结果尿激酶溶凝作用随时间延长逐渐降低,不同尿激酶剂量在加药后1、2、3、4h均有显著性差异（P〈0．05）;4h后无明显差异（P〉0．05）。各时间点,0．5、1、2万U尿激酶无显著性差异（P〉O．05）,5、10及15万U元显著性差异（P〉0．05）,但0、5、1、2万U和5、10、15万u间均有显著性差异（P〈0．05）。结论本研究提示尿激酶溶凝最佳作用时间为4h,尿激酶首次最佳用量为1万U／10ml。     

Comparative analysis of fibrinolytic properties of Alteplase,Tenecteplase and Urokinase in an in vitro clot model of intracerebral haemorrhage

ObjectiveHematoma lysis with recombinant tissue plasminogen activator (rtPA) has emerged as an alternative therapy for spontaneous intracerebral and intraventricular haemorrhage (ICH and IVH). However, the MISTIE III and CLEAR III trial failed to show significant improvement of favourable outcomes. Besides experimental and clinical trials revealed neurotoxic effects of rtPA. The demand for optimization of fibrinolytic therapy persists. Herein, we used our recently devised clot model of ICH to systematically analyse fibrinolytic properties of rtPA, tenecteplase and urokinase.MethodsIn vitro clots of human blood (size: 25 ml and 50 ml; age: 1.5 tenecteplase, 24 tenecteplase and 48 tenecteplase) were produced and equipped with a catheter into the clot core for drug delivery and drainage. Various doses of tenecteplase and urokinase with different treatment periods were examined (overall 117 clots), assessing the optimal dose and treatment time of these fibrinolytics. Clots were weighed before and at the end of treatment. These results were compared with clots treated with 1 mg rtPA or with 0.9% sodium chloride solution.ResultsThe optimal treatment scheme of tenecteplase was found to be 100 IU with an incubation time of 30 min, for urokinase it was 50 000 IU with an incubation time of 20 min. The relative clot end weight of tenecteplase and urokinase (31.3±11.9%, 34.8 ±7.7%) was comparable to rtPA (36.7±10.7%). Larger clots were more effectively treated with tenecteplase compared to the control group (P=0.0013). urokinase and tenecteplase had similar lysis rates in aged clots and 90 min clots. One and two repetitive treatments with tenecteplase were as effective as two and three cycles of urokinase.ConclusionsIn our in vitro clot model we could determine optimal treatment regimens of tenecteplase (100 IU, 30 min) and urokinase (50 000 IU, 20 min). Urokinase and tenecteplase were comparable in their fibrinolytic potential compared to 1mg rtPA in small clots and showed an effective lysis in aged clots. tenecteplase was more effective in larger clots.     

Correlation between progressive adsorption of plasminogen to blood clots and their sensitivity to lysis

The binding of plasminogen to preformed human plasma clots immersed in citrated human plasma was measured and correlated with the sensitivity of these clots to lysis with recombinant tissue-type plasminogen activator (rt-PA), recombinant single-chain urokinase-type plasminogen activator (rscu-PA) or two chain urokinase-type plasminogen activator (tcu-PA, urokinase). When 0.15 ml plasma clots were compressed mechanically to about 1% of their original weight, and immersed in 0.15 ml plasma, 131I-labeled native plasminogen (Glu-plasminogen) adsorbed progressively from the plasma milieu onto the clot; binding was 3 +/- 1% (n = 10) after 1 h, 7 +/- 1% after 12 h and 12 +/- 1% after 48 h. This was associated with an increased sensitivity of the clot to lysis; 50% clot lysis in 4 h was obtained with 65 +/- 5 ng/ml (n = 3) rt-PA before and 30 +/- 5 ng/ml (n = 3) after 48 h preincubation in plasma (p less than 0.01), with corresponding values of 660 +/- 55 ng/ml (n = 3) and 280 +/- 25 ng/ml (n = 3) for rscu-PA, (p less than 0.01), and 800 +/- 85 ng/ml (n = 3) and 270 +/- 35 ng/ml (n = 3) for urokinase (p less than 0.01). Additional binding of plasminogen and increased sensitivity to lysis were reduced or abolished when the clot was preincubated in plasminogen-depleted or in t-PA-depleted plasma, or when 20 mM 6-aminohexanoic acid or 2,000 KIU/ml aprotinin were added.(ABSTRACT TRUNCATED AT 250 WORDS)     

Turbulent axially directed flow of plasma containing rt-PA promotes thrombolysis of non-occlusive whole blood clots in vitro

The rate of thrombolysis markedly decreases after a thrombosed vessel is partly recanalized and the remaining clot poses serious risk for rethrombosis. We studied in vitro how thrombolysis depends on penetration of plasma containing thrombolytic agents - 0.2 micro g/ml rt-PA or 250 IU/ml streptokinase (SK) - and the magnetic resonance contrast agent Gd-DTPA (at 1 mmol/l) into non-occlusive clots under conditions of fast (turbulent) or slow (laminar) axially directed flow. Cylindrical non-retracted (fresh) or retracted (aged) whole blood clots were pierced lengthways and connected to a perfusion system. Dynamical spin-echo MRI was used for measuring the penetration of labeled plasma into clots and for assessing the remaining clot size. In both types of clots fast flow enhanced the penetration of Gd-DTPA-labeled plasma into clots in comparison to slow flow. In non-retracted clots, lysis with rt-PA and to a lesser extent also lysis with SK followed the path of plasma penetration into clots. After 40 minutes of fast axially directed flow rt-PA resulted in almost complete lysis and SK left only about a third of the clot undissolved, whereas with slow flow lysis was much slower (undissolved clot: 86 +/- 5 % with rt-PA and 95 +/- 1 % with SK). In retracted clots, substantial lysis was possible only with rt-PA and rapid flow (53 +/- 28% of the clot undissolved after 60 min), whereas the use of SK or slow flow precluded meaningful lysis. We conclude that rapid (turbulent) axially directed flow of plasma along non-occlusive blood clots causes forceful exchange of serum inside the clot with outer plasma which enhances both fibrin-specific and non-fibrin-specific lysis of fresh clots. Dissolution of non-occlusive retracted (aged) clots occurs only under fibrin-specific conditions combined with adequate transport of rt-PA into clots.     

Clot penetration and fibrin binding of amediplase,a chimeric plasminogen activator (K2 tu-PA)

Amediplase (K(2) tu-PA) is a hybrid plasminogen activator, consisting of the kringle 2 domain of alteplase and the protease domain of urokinase. The objective of this study was to determine the in vitro clot penetration of amediplase in relation to its fibrin binding and to compare the properties with those of alteplase. The clot lysis activity of amediplase in internal clot lysis models (both purified system and plasma system) was about 10 times less than that of alteplase. The clot lysis activity of amediplase in an external clot lysis model (plasma system) was similar to that of alteplase at therapeutic concentrations around 1 micro g/ml. The fibrin-clot binding properties of amediplase and alteplase were studied in a purified system as well as in a plasma system. In both systems amediplase bound to fibrin although to a significantly lower extent than alteplase. The binding of amediplase or alteplase did not increase during plasmin-mediated degradation of fibrin. The binding of amediplase was fully inhibited by epsilon-aminocaproic acid, indicating that the observed binding was specific and occurred via the lysine binding site in the kringle of amediplase. Clot penetration was studied during pressure-driven fluid permeation using syringes containing plasma clots. Amediplase was able to enter the clot without significant hindrance, while alteplase was concentrated on the top of the plasma clot and hardly entered into the inner parts of the clot. Diffusion-driven clot penetration was studied during clot lysis using confocal microscopy. Alteplase was detected on or close to the clot surface, while two-chain urokinase, which has no affinity to fibrin, was also detected deep inside the clot. Amediplase showed a penetration behaviour, which was distinct from that of alteplase and similar to that of two-chain urokinase. We concluded that the fibrin binding of amediplase is moderate and does not hinder clot penetration under permeation-driven or diffusion-driven transport conditions. Enhanced clot penetration, especially in large clots, could allow a more efficient lysis during thrombolytic therapy.     

The influence of transport parameters and enzyme kinetics of the fibrinolytic system on thrombolysis: mathematical modelling of two idealised cases.

Experimental data obtained by magnetic resonance imaging and photographing clot dissolution in vitro have shown that whole blood clots dissolve almost two orders of magnitude faster when urokinase is introduced into the clot by pressure induced permeation than when its access is limited to diffusion. In view of these findings, two mathematical models have been developed that quantitatively link the enzymatic and transport properties of the fibrinolytic system to the velocity of thrombolysis. Without a pressure gradient across the thrombus, the plasminogen activator molecules diffuse into the thrombus through the blood-thrombus boundary plane. The blood-thrombus boundary slowly moves inwards due to thrombolysis that is spatially restricted to a relatively narrow zone. The velocity of thrombolysis is primarily limited by the diffusion constants of the plasminogen activator and plasmin. In contrast, when plasminogen activator is rapidly distributed along the thrombus by pressure induced bulk flow, lysis occurs at each segment of the thrombus after a lag period that is due to plasmin activation and sufficient fibrin degradation. The lag time is determined primarily by the catalytical properties of the plasminogen activator and plasmin. The mathematical models with the observations of the clot boundaries during lysis permit the characterization of plasmin action on the fibrin network.     

Stimulation of the plasma fibrinolytic activity in rats by the prostacyclin analogue CG 4203

In anesthetized rats the intravenous infusion (15-120 min) of the prostacyclin analogue CG 4203 (0.215-2.15 micrograms.kg-1.min-1) resulted in a time and dose dependent shortening of the ex vivo euglobulin clot lysis time (ECLT). This effect that appeared to be significant already in the non-hypotensive dose range of CG 4203, was still existing at 2 hours after cessation of the infusion. The phosphodiesterase inhibitor theophylline (4.64 mg.kg-1 i.v.) potentiated the ECLT shortening effect of CG 4203. Even the highest dose of CG 4203 did not change the plasma fibrinogen levels. In contrast to low molecular weight urokinase (100 PU/ml) CG 4203 (10 microM) did not shorten the in vitro lysis of preformed euglobulin clots from untreated rats nor did it reduce the 125J-fibrin content of human thrombi in the Chandler loop system. From these results it is concluded that intravenously infused CG 4203 increases the plasma fibrinolytic activity in rats by a c-AMP dependent mechanism, probably by release of plasminogen activator. Direct urokinase like activation of plasminogen does not occur with CG 4203. The relevance of this activity is discussed with respect to the CG 4203 treatment of occlusive vascular diseases.     

Influence of exogenous and endogenous tissue-type plasminogen activator on the lysability of clots in a plasma milieu in vitro

The fibrinolytic potential of tissue-type plasminogen activator (t-PA) either incorporated in a clot (endogenous) or added to the surrounding plasma (exogenous), was studied in an in vitro system consisting of 125I-labeled human plasma clots (200 microliters) immersed in human plasma (2 ml). Clot lysis was measured as a function of endogenous t-PA concentration (in the absence of added exogenous t-PA), as a function of exogenous t-PA concentration (without added endogenous t-PA) and as a function of the same concentration of both endogenous and exogenous t-PA. Equivalent clot lysis was obtained with a 2 to 4 times lower concentration of endogenous t-PA as compared to exogenous t-PA, corresponding to a 20 to 40 times smaller total amount of endogenous versus exogenous t-PA. Fifty percent lysis in 5 hrs was obtained with about 5 IU/ml of endogenous t-PA or with 10 IU/ml of exogenous t-PA. The presence of both exogenous (10 IU/ml) and endogenous (5 IU/ml) t-PA resulted in 50 percent lysis in 1.5 hrs, indicating that t-PA incorporated in a thrombus contributes significantly to its lysis by exogenous t-PA. Similar results were obtained with plasma obtained after 10 min of venous occlusion in seven healthy subjects. Spontaneous clot lysis within 5 hrs was only observed with post-occlusion clots in pre- or post- occlusion plasma in two subjects in whom the t-PA level rose to 10-15 IU t-PA/ml. In the five other subjects with post-occlusion t-PA levels below 2 IU/ml, no clot lysis was observed within 24 hrs. The influence of the fast-acting inhibitor of t-PA on clot lysability by endogenous or exogenous t-PA was investigated by immersing clots prepared from normal or inhibitor-rich plasma (endogenous inhibitor) in normal or inhibitor-rich plasma (exogenous inhibitor). Exogenous t-PA inhibitor efficiently neutralizes clot lysis by both exogenous and endogenous t-PA. Endogenous t-PA inhibitor, however, efficiently neutralizes endogenous t-PA but has little influence on clot lysis by exogenous t-PA. These findings indicate that t-PA inhibitor is not concentrated into a clot and that t-PA inhibitor in plasma efficiently neutralizes t-PA incorporated in a clot. alpha 2-Antiplasmin depleted plasma clots were more susceptible to lysis by both endogenous and exogenous t-PA than normal clots.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro studies on the fibrinolytic, thrombolytic and fibrinogenolytic properties of a tissue plasminogen activator from guinea pig keratocytes

The fibrinolytic and thrombolytic properties of a tissue plasminogen activator (tPA) purified from the conditioned medium of an established guinea pig keratocyte (GPK) cell line were investigated in in vitro systems and compared with urokinase. Using the fibrin clot lysis assay, GPK activator appears to be similar to human melanoma tPA and not to human urokinase. GPK activator also caused negligible fibrinogen breakdown, when incubated with human plasma at 37 degrees C over 23 hr. Urokinase on the other hand caused significant fibrinogenolysis, under similar conditions. Comparison of the lysis of plasma clots by GPK activator and human urokinase have shown that GPK activator was a much more effective fibrinolytic agent than urokinase, especially at lower concentrations (less than 50 IU/ml). Studies on the thrombolytic effect of GPK activator on the lysis of aged and cross-linked whole human blood clots and plasma clots hanging in artificially circulating human plasma suggest that GPK activator can lyse both these types of clots equally well. The lysis is dose dependent, attaining complete lysis within 3-6 hr with the concentration of GPK activator in the range of 1-5 micrograms/ml plasma. It is concluded that GPK activator has a higher fibrinolytic and thrombolytic activity and lower fibrinogenolytic activity than urokinase.     

Biological and thrombolytic properties of proenzyme and active forms of human urokinase--IV. Variability in fibrinolytic response of plasma of several mammalian species

The fibrinolytic and fibrinogenolytic properties of recombinant pro-urokinase (Rec-pro-UK) and recombinant urokinase (Rec-UK) were compared with those of natural urokinase (Nat-UK) and of tissue-type plasminogen activator (t-PA) in an in vitro system consisting of 125I-labeled autologous plasma clots immersed in plasma of humans, five primate species, dogs, rabbits and pigs. With each of the four plasminogen activators, a dose-dependent clot lysis was observed, the degree of which differed, however, very markedly from one species to the other. At a concentration of 100 IU/ml of urokinase extensive plasma clot lysis was obtained in plasma of man, Macaca mulatta, Macaca fascicularis and Macaca radiata, while the plasma clots of Papio cynocephalus, Papio anubis and rabbit, dog and pig were much more resistant to lysis. No significant differences in the extent of lysis were observed between Rec-pro-UK and Rec-UK nor between Rec-UK and Nat-UK. Comparable degrees of lysis were obtained with t-PA at 3- to 5-fold lower concentrations. Lysis with Rec-UK or Nat-UK was always associated with extensive activation of the fibrinolytic system in plasma, evidenced by fibrinogen breakdown and plasminogen activation and alpha 2-antiplasmin consumption. With t-PA, extensive clot lysis was obtained in the absence of fibrinolytic activation in the plasma. With Rec-pro-UK the response was intermediate; at high concentrations (200 IU/ml) extensive lysis in the reactive species was associated with fibrinogen consumption, while at intermediate concentrations (50-100 IU/ml) significant clot lysis was obtained in the reactive species in the absence of marked activation of the fibrinolytic system in the plasma.     

The effect of flow on lysis of plasma clots in a plasma environment

Fibrinolysis initially generates channels in an occluding thombus which results in blood flow through the thrombus. Since the impact of flow along the surface of a thrombus on thrombolysis has not been investigated in detail, we studied in vitro how such a flow affects lysis. Compacted and noncompacted plasma clots were used as model thrombi. With compacted clots, fibrin-specific lysis induced by alteplase in the outer plasma was accelerated about 2-fold by strong flow (arterial shear rate). Non-fibrin-specific lysis induced either by a high concentration of alteplase or by streptokinase was slow, was accompanied by rapid depletion of plasminogen in the outer plasma, and was only slightly accelerated by flow. With noncompacted clots, similar acceleration factors were documented, when mild flow (venous shear rate) was applied. Strong flow further accelerated fibrin-specific lysis, up to 10-fold as compared to lysis without flow, but paradoxically retarded non-fibrin-specific lysis. The data suggest that flow accelerates lysis by enhancing transport of plasminogen from the outer plasma to the surface of the clot. Both opposite effects of the strong flow were mediated by forceful intrusion of the outer plasma into the noncompacted clot due to flow irregularities. In the case of non-fibrin-specific lysis this resulted in the replacement of the plasminogen-containing milieu by plasminogen-depleted outer plasma in certain areas of the clot turning them into virtually unlysable fragments. This flow-enforced "plasminogen steal" may contribute to the relatively high percentage of incomplete thrombolysis (TIMI-2 grade flow) documented in a number of trials for non-fibrin-specific thrombolytic agents. In the case of fibrin-specific lysis, the effect of flow on the speed of fibrinolysis is always beneficial.     

Specific fibrinolytic properties of different molecular forms of pro-urokinase from a monkey kidney cell culture

The specific fibrinolytic properties of both high molecular weight (55 kd) and low molecular weight (30 kd) pro-urokinase from a monkey kidney cell culture were evaluated in a plasma clot lysis system and compared with those of human urokinase. The system was composed of a radiolabelled plasma clot immersed in plasma containing the fibrinolytic agent. On unit base, 55 kd pro-urokinase was approximately 1.5 times more effective in lysing the clot than 30 kd pro-urokinase and equally effective as urokinase. In contrast to urokinase, both pro-urokinase forms induced clot lysis without degrading fibrinogen in the surrounding plasma. However, a considerable activation of the fibrinolytic system in the plasma occurred as a large amount of ₂-antiplasmin was consumed, indicating that pro-urokinase was not fully fibrin-specific. Quenching antibodies against tissue-type plasminogen activator (t-PA) added to the plasma clot lysis system retarded but did not prevent pro-urokinase-induced clot lysis. This indicated that not only was t-PA in plasma involved in the activation of pro-urokinase (probably via plasmin), but that an additional mechanism also existed.     

Induction of a sustained fibrinolytic response by BRL 26921 in vitro

The role of thrombus-binding in the fibrinolytic response to the acylated streptokinase.plasminogen activator complex, BRL 26921, has been examined using human plasma clots, radiolabelled with 125I-fibrin, in vitro. When clots were briefly exposed to BRL 26921, washed and returned to homologous plasma, lysis continued for up to 3 hours and attained approximately 25% of that lysis achieved by incubating with BRL 26921 for 5 hours. This continuing lysis was potentiated by return of exposed clots to alpha 2-antiplasmin-depleted plasma, or buffer and is attributed to an initial uptake of BRL 26921 rather than the binding of exogenous plasmin that was observed for streptokinase and high concentrations of urokinase. The sustained lysis is not explained by transfer of loosely-associated surface material or by dissociation of agent from the clot with reuptake from a dilute systemic pool. The response can be attributed, at least in part, to specific fibrin binding, mediated by kringles 1-4, for a low-molecular weight plasminogen (Val442) variant was less active.     

Comparison of affinities of urokinase and tissue plasminogen activator for fibrin clots

Affinities of low molecular weight two-chain urokinase (UK) and tissue plasminogen activator (t-PA) for fibrin clots were investigated by using clot lysis rates to estimate an affinity (Kd) between activator and fibrin clots. Lysis rates were obtained using a simple spectrophotometric based clot lysis assay which is described here. Fibrin clots, containing residual plasminogen, were suspended in a 1 ml cuvette and the increase in absorbance at 280 nm due to release of soluble fibrin peptides measured over a 150 to 250 minute time period. Lysis rates were obtained from plots of time squared vs absorbance change. Plots of activator concentration vs reciprocal rates yielded regression coefficients of 0.999 and Kd values of nM for the affinity of both activators for fibrin clots. Although both activators are known to differ in affinity for fibrin, they nonetheless had similar affinities and lysis rates for the insoluble fibrin clots. This assay also suggested possible synergism; rates over twice that expected by an additive effect were observed when the two activators were mixed at 0.3 to 7.6 nM each.     

Factor XIII cross-linking and the rate of fibrinolysis induced by streptokinase and urokinase

The rate of lysis induced by streptokinase or urokinase of fibrin clots with varying degrees of Factor XIII cross-linking was investigated. Various techniques were used in which the plasminogen activator was uniformly distributed through the clot, or was present only at the surface. The clots were produced from purified fibrinogen solutions or from plasma. None of these ivestigations gave any evidence to indicate that lytic rates were decreased by the cross-linking of either the α or γ chains of fibrin.     

In vivo penetration of experimentally produced clots by monoclonal antibodies

Antifibrin monoclonal antibodies show potential as clot targeting agents for diagnosis and possibly therapy in thrombotic disease. To be effective the antibody must bind to the fibrin component of the clot. The ability of two antifibrin mabs (NIB 1H10 and NIB 12B3) to penetrate occlusive clots in vivo was investigated. Both mabs react with human fibrin but not with human fibrinogen nor with the fibrin or fibrinogen from the species used in this study. Two heterologous animal (sheep and rabbit) thrombus models were used. Clots in both cases were made within isolated vein segments using a mixture of human and native fibrinogen. The clots in sheep veins were observed radiographically and found to be occlusive for a mean of 4.2 +/- 2.2 days and thereafter appeared only partially occlusive. When targeted in their occlusive phase (131)I labelled mab accumulated in the clot reaching a maximum ratio of 1.82 +/- 0.42 when compared to counts in homologous sheep clots in the contralateral limb. It was confirmed in the rabbit jugular vein model that total occlusivity did not prevent antibody accumulation in the heterologous clot by injecting the fibrin specific mab 1H10 and examining the clot excised after 1, 6 and 24 h using immunofluorescence. In a further series of similar experiments (125)I labelled mab 1H10 was used and detected using autoradiography. Both sets of experiments indicated that penetration of occlusive clots by the antibody occurred and that considerable accumulation was present at 6 and 24 h. The results indicate that a circulating antibody can readily gain access to experimentally produced clots in occluded veins.     

大鼠脑室出血鞘内与脑室内纤溶治疗对比研究

目的研究大鼠鞘内与脑室内注射尿激酶两种纤溶治疗对脑室系统血凝块的影响。方法借助大鼠脑立体定位仪建立大鼠脑室出血模型；测定腩组织含水量，并比较不同途径纤溶治疗对脑组织水肿的影响；通过脑片及HE染色观察血凝块清除情况。结果两治疗组鞘内组和脑室组脑组织含水量较模型组明显减少（P〈0．05），但鞘内组和脑室组之间脑组织含水量无显著性差异（P〉0．05）；脑片及HE染色观察到模型大鼠脑室系统内血凝块有残留，而两治疗组脑室血块大部分被清除。结论脑室出血后经鞘内和脑室内纤溶治疗均能加快血块溶解，促进脑脊液循环，减轻脑组织水肿和损伤。     

Factor XIIa is a kinetically favorable plasminogen activator

Initiation of the plasma contact system has been shown to play a significant role in the fibrinolysis, activating both pro-urokinase and plasminogen. The aim of the present study was to further evaluate the functional role of the factor XIIa catalyzed activation of plasminogen. Activation of plasminogen by factor XIIa followed the Michaelis-Menten rate equation. In a continuous assay system the Km was 0.27 microM; the kcat 0.078 min(-1) and the kcat/Km 0.31x10(6) M(-1) x min(-1). In an end-point assay system the Km was 0.58 microM; the kcat 0.096 min(-1) and the kcat/Km 0.16x10(6) M(-1) x min(-1). The discrepancy between the kcat in the two assays is not significant. Theoretically the higher Km in the end-point assay system may be due to the presence or generation of an unidentified competitive inhibitor in this assay system. Comparing the catalytic constants of factor XIIa with those of urokinase activation of plasminogen, the specificity constant, kcat/Km, of factor XIIa activation of plasminogen was 20-fold lower. However, taking the low physiological concentration of urokinase into account, the efficiency of activated factor XII is equivalent to that of urokinase. When monitoring factor XIIa activation of plasminogen in a clot lysis assay, the clot lysis time was 2- to 4-fold as long as that accommodated by urokinase at factor XIIa concentrations equivalent with 5-20% of the zymogen concentration in plasma. The factor XIIa mediated clot lysis was prevented completely by the presence of a polyclonal antibody to factor XII.     

治疗用超声辅助尿激酶溶栓作用的体外研究

目的 在体外环境下,评估治疗用超声溶栓作用以及影响尿激酶溶栓效率的因素.方法 从血液离体后分别在3、4、5、6、7、8、9、10 h 8个时间点制备离体血栓并开始溶栓,分为生理盐水组(NS),尿激酶组(UK),生理盐水+超声组(NS+US),尿激酶+超声组(UK+US),每组32份血栓,放入37 ℃的水浴环境下干预1 h后再称重,计算血栓失重和溶栓率并观察离体血栓存放时间对尿激酶溶栓效果的影响.结果 UK组和UK+US组的溶栓率分别是29.3%±8.2%、37.5%±7.9%,两者比较LSD-t值为12.1,P＜0.01,差异有统计学意义,NS组和NS+US组的溶栓率分别是13.4%±4.4%、14.5%±5.4%,两者相比较LSD-t值为1.8,P值为0.08,差异无统计学意义;在一定范围内,离体血栓存放时间越长,溶栓率越低.结论 在体外环境下,治疗用超声可能本身并无溶栓作用,但可以明显增强尿激酶的溶栓作用;在一定范围内,离体血栓存放时间越长则溶栓率越低.