降纤酶治疗急性脑梗死的临床研究

目的　评估降纤酶对急性脑梗死的疗效及安全性。　方法　采用前瞻性、随机、对照试验 ,以东菱精纯克栓酶 ( DF-52 1 )和低分子右旋糖酐为对照组 ,从 1 996年 1月至 1 998年 1 0月共观察 4 81例 ,其中降纤酶组 2 89例 ,东菱组 92例 ,低右组 1 0 0例 ,共完成试验者 30 1例 ,各组份别为 1 4 9例、 70例和 82例 ,于治疗前及治疗后第 1天 ,第 3天 ,第 7天 ,第 1 4天查血浆纤维蛋白原、凝血酶原时间及血小板计数、肝肾功能等。并于治疗前及治疗后 1 ,7,1 4天评估神经功能恢复情况。　结果　降纤酶对急性脑梗死疗效显著 ,明显优于低分子右旋糖酐 ,在治疗后 1天已表现出明显效果 ,与日本生产的东菱精纯克栓酶比较疗效相似 ,发病 1 4天治疗总有效率分别为 84 .4 5%和 80 .0 0 %。同时降纤酶组与东菱组治疗后纤维蛋白原下降明显 ,于 7～ 1 4天有回升趋势 ;低右组则无明显变化。　结论　降纤酶具有与东菱精纯克栓酶相似的疗效、副作用小 ,价格明显低于东菱精纯克栓酶。认为国产降纤酶可以取代东菱精纯克栓酶 ,其在脑梗死急性期治疗上具有广泛的前景     

Collagen-bound von Willebrand factor has reduced affinity for factor VIII

von Willebrand factor (vWf) is a multimeric adhesive glycoprotein that serves as a carrier for factor VIII in plasma. Although each vWf subunit displays a high affinity binding site for factor VIII in vitro, in plasma, only 2% of the vWf sites for factor VIII are occupied. We investigated whether interaction of plasma proteins with vWf or adhesion of vWf to collagen may alter the affinity or availability of factor VIII-binding sites on vWf. When vWf was immobilized on agarose-linked monoclonal antibody, factor VIII bound to vWf with high affinity, and neither the affinity nor binding site availability was influenced by the presence of 50% plasma. Therefore, plasma proteins do not alter the affinity or availability of factor VIII-binding sites. In contrast, when vWf was immobilized on agarose-linked collagen, its affinity for factor VIII was reduced 4-fold, with KD increasing from 0.9 to 3.8 nM. However, one factor VIII-binding site remained available on each vWf subunit. A comparable reduction in affinity for factor VIII was observed when vWf was a constituent of the subendothelial cell matrix and when it was bound to purified type VI collagen. In parallel with the decreased affinity for factor VIII, collagen-bound vWf displayed a 6-fold lower affinity for monoclonal antibody W5-6A, with an epitope composed of residues 78-96 within the factor VIII-binding motif of vWf. We conclude that collagen induces a conformational change within the factor VIII-binding motif of vWf that lowers the affinity for factor VIII.     

Double cryoprecipitated factor VIII concentrate from heparinised plasma and its heat treatment

In an attempt to implement the small pool concept in Factor VIII purification, cryoprecipitate derived from heparinised plasma was reprecipitated in the cold providing a factor VIII concentrate for freeze drying and heat treatment. There was considerable purification; only 1% of the original plasma proteins was left in the final product. Factor VIII:C concentration was about 19 IU/ml. Factor VIII related antigen (RAg) appeared heterogeneous, with a broad base and asymmetry on crossed immunoelectrophoresis. Fibrinogen content was 15 g/l. In contrast to high-purity commercial concentrates, fibronectin was considerably concentrated. Immunoglobulin contents were similar to a high-purity commercial product. The amount of other plasma proteins was very small, varying from less than 0.2% for C3 complement to 2.3% ceruloplasmin. In some respects the preparation may be considered as an intermediate-purity Factor VIII concentrate. Following addition of 2% sucrose before freeze drying, Factor VIII, total protein and fibrinogen remain virtually stable (less than 15% loss) during heating of the material to 60, 64 or 68 degrees C for 24 to 72 h without changes of protein spectrum following heating. The heated product when stored at 4 degrees C remains stable for at least 3 months. In two severe haemophiliacs receiving this heat treated product, in vivo Factor VIII recovery was 100% with a mean half life of 10.2 h.     

Peptide map analysis of normal plasma and platelet factor VIII antigen

Factor VIII antigen from platelet intracellular granules was immunoprecipitated using a monospecific rabbit antibody to normal plasma factor VIII antigen. The factor VIII antigen in the immunoprecipitate was isolated on sodium dodecyl sulfate polyacrylamide gels, radiolabeled with ¹²⁵I, trypsinized, and subjected to peptide mapping using two dimensional high voltage electrophoresis and thin layer chromatography. The platelet protein was compared to purified plasma factor VIII antigen. The two dimensional tryptic ¹²⁵I peptide map of platelet granule factor VIII antigen was similar but not identical to the plasma factor VIII antigen peptide map. The platelet and plasma proteins shared approximately 34 radioactive peptide spots. Seven plasma factor VIII antigen peptides were not detected in platelet factor VIII antigen. The reason for the structural differences of plasma and platelet granule factor VIII antigen are unknown. The possibility is raised that proteolysis has altered the platelet protein in vitro. It is also possible that factor VIII antigen synthesized by megakaryocytes differs from the plasma protein.     

Specificity of phosphatidylserine-containing membrane binding sites for factor VIII. Studies with model membranes supported by glass microspheres (lipospheres).

Factor VIII functions in an enzyme complex upon the activated platelet membrane where phosphatidylserine exposure correlates with expression of receptors for factor VIII. To evaluate the specificity of phosphatidylserine-containing membrane binding sites for factor VIII, we have developed a novel membrane model in which phospholipid bilayers are supported by glass microspheres (lipospheres). The binding of fluorescein-labeled factor VIII to lipospheres with membranes of 15% phosphatidylserine was equivalent to binding to phospholipid vesicles (KD = 4.8 nM). Purified von Willebrand factor (vWf), a carrier protein for factor VIII, decreased membrane binding of factor VIII with a Ki of 10 micrograms/ml. Likewise, normal plasma decreased bound factor VIII by more than 90% whereas plasma lacking vWf decreased the binding of factor VIII by only 20%. Proteolytic activation of factor VIII by thrombin, which releases factor VIII from vWf, increased liposphere binding in the presence of vWf and in the presence of normal plasma. Although factor V is homologous to factor VIII and binds to lipospheres with the same affinity, purified factor V was not an efficient competitor for the membrane binding sites of factor VIII. These results indicate that phosphatidylserine-containing membrane sites have sufficient specificity to select thrombin-activated factor VIII from the range of phospholipid-binding proteins in plasma.     

Coagulation factor levels in non-metastatic colorectal cancer patients

There is evidence that high plasma levels of factor (F) VIII, FIX, FXI and fibrinogen are independent risk factors for venous thromboembolism. AIM: To determine the plasma concentrations of several coagulation factors and C4b-binding protein (C4BP) in a group of patients with non-metastatic colorectal cancer in order to investigate some aspects of cancer-acquired thrombophilia. METHODS: Plasma fibrinogen, FII, FV, FVII, FVIII, FIX, FX, FXI and FXII activity levels and C4BP concentrations were determined in 73 patients with non-metastatic colorectal cancer (48 colon and 25 rectum) and in 67 matched control subjects. No one in either group had had previous thrombotic events. RESULTS: Mean plasma concentrations of fibrinogen (functional and antigen), FVIII, FIX, FV and C4BP were significantly higher in colorectal cancer patients than in control subjects, while FVII and FXII levels were significantly decreased. Several correlations were found between the increased coagulation factors and C4BP concentrations, while FVII was highly correlated with FXII. CONCLUSIONS: In colorectal cancer patients high plasma fibrinogen, FVIII and FIX levels might represent further risk factors for venous thrombotic complications in the immediate post-surgery period, while decreased FVII and FXII concentrations may be an index of intravascular coagulation activation, still in a subclinical phase.     

Plasma exchange and human factor VIII concentrate in managing haemophilia A with factor VIII inhibitors.

Plasma exchanges were combined with human factor VIII concentrate therapy in the treatment of major bleeding episodes in five patients with haemophilia A and factor VIII inhibitors. All patients had a good clinical response to combined treatment. Inhibitor levels showed satisfactory falls before rapid secondary increases of inhibitor levels took place. A sixth patient with von Willebrand''s disease and a factor VIII clotting activity inhibitor was successfully prepared for operation using plasma exchange. Postoperative haemostasis and healing were normal. In two patients the plasma exchanges were relatively more effective than the administered human factor VIII in reducing the levels of factor VIII inhibitor. Combined plasma exchange and human factor VIII treatment may offer a rapidly effective means of reducing factor VIII inhibitor levels in this group of patients, together with significant saving of costs.     

Multiple molecular forms of endothelial cell factor VIII related antigen

Heterogeneous factor VIII related antigen isolated from endothelial cell postculture medium was characterized. On crossed immunoelectrophoresis, slow moving less anodal populations of factor VIII related antigen molecules were more prominent in endothelial cell postculture medium than in plasma. The protein synthesized by endothelial cells appeared as two discrete protein bands of different molecular weight in sodium dodecyl sulfate polyacrylamide gels. In contrast the factor VIII related antigen isolated from plasma moved as a single protein band in an identical gel system. The factor VIII related antigen from endothelial cell postculture medium was immunoisolated using monospecific rabbit antibody to normal human plasma factor VIII related antigen, electrophoresed on sodium dodecyl sulfate polyacrylamide gels, radiolabeled with ¹²⁵I, trypsinized and subjected to peptide mapping using two-dimensional high voltage electrophoresis and thin-layer chromatography. The two forms of endothelial cell factor VIII antigen had virtually identical peptide maps. These studies suggest that the molecular heterogeneity of the factor VIII related antigen sytem reflects polymeric associations of identical subunits. Circulation in vivo may alter the ratio of polymer subsets.     

The interaction of the factor VIII/von Willebrand factor complex (VIII/vWf), with guanidinium-derivatized matrices.

Five different guanidinium (Gu)-derivatized agarose matrices were investigated for their potential in chromatographically resolving the Factor VIII/von Willebrand complex, VIII/vWf, fibrinogen, Fg, and fibronectin, Fn, from cryoprecipitate. Using conventional NaCl gradient methodology it was found that the order of elution of specific plasma proteins, and the yield of VIII/vWf, varied with the methods used to derivatize the agarose beads. Good yields of VIII:C (generally 30-45%) were obtained with Gu-matrices prepared by bis-oxirane coupling procedures. Cryoprecipitate binding studies showed that the capacity of Gu-Sepharose 4B, prepared by isourea modification of amino-Sepharose 4B, was 36 units VIII/vWf per ml matrix. The product, depleted of both Fg and Fn, had a specific activity of 2 units VIII:C per mg total protein, (yield 100% vWf:Ag and 47% VIII:C).     

Separation of human plasma fibrin stabilizing factor (factor XIII) from fibrinogen by fibrinogen polymer formation

Human plasma fibrin stabilizing factor (factor XIII) may be separated from fibrinogen through reversible fibrinogen polymer formation at pH 6.6, gamma/2 0.3, 0 degrees C, and subsequent Bio-Gel A 1.5m filtration. Factor XIII activity is eluted after the monomer fibrinogen peak. Polymer fractions from eight preparations, processed in duplicate, contain a mean 0.002 units factor XIII per mg fibrinogen, or about 0.7% the factor XIII content of standard plasma. Factor XIII-free fibrinogen polymers are easily dissociated (greater than 98%) to the monomer form by incubation at 37 degrees C, 18 hours. The fibrinogen preparations utilized were devoid of plasma fibronectin; thus these studies also show that reversible human fibrinogen polymer formation occurs in its absence.     

Stoichiometry of the porcine factor VIII-von Willebrand factor association

Factor VIII and von Willebrand factor (vWF) are glycoproteins that form a tightly bound complex in plasma. The interaction of porcine factor VIII with porcine vWF was studied by analytical velocity sedimentation. A single approximately 240-kDa species of factor VIII was isolated for use in the analysis. In contrast, when analyzed by agarose/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, vWF consisted of a population of greater than 10 multimers derived from a 270-kDa monomer. A single boundary (So20,w = 7.2 S) was observed during velocity sedimentation of factor VIII at 260,000 x g. A single boundary also was observed for vWF (weight-average So20,w = 21 S) at 42,000 x g. Under condition of excess factor VIII, the weight-average So20,w of the factor VIII-vWF complex was 40 S at 42,000 x g. At 260,000 x g, the factor VIII-vWF complex had sedimented completely, leaving only free factor VIII. The height of the plateau region of the factor VIII sedimentation velocity curve at 260,000 x g was studied as a function of several starting concentrations of vWF. The experiments were done under conditions in which the effect of radial dilution was negligible so that the plateau height was a measure of the concentration of free factor VIII. The plateau height decreased linearly as the concentration of vWF was increased, indicating that the association was essentially irreversible under the conditions used. A stoichiometry of 1.2 vWF monomers/factor VIII molecule was calculated from the slope of the line. Assuming one factor VIII-binding site/vWF monomer, these results indicate that all factor VIII-binding sites are accessible in the vWF multimer.     

The role of von Willebrand factor multimers and propeptide cleavage in binding and stabilization of factor VIII

von Willebrand factor (vWF) is a multimeric glycoprotein that promotes platelet aggregation and stabilizes coagulation factor VIII in the plasma. vWF is also required for the stable accumulation of recombinant factor VIII secreted from cells in a heterologous expression system. In this report, we show that vWF can promote the in vitro reconstitution of factor VIII activity from dissociated heavy and light chains of factor VIII, suggesting that vWF may act to promote stable assembly of factor VIII subunits at the site of secretion. The structural requirements for vWF propeptide cleavage and for vWF multimerization in its binding and stabilization of factor VIII was examined using specifically altered recombinant vWF. The mutant vWF molecules were also assayed for their function in ristocetin-induced platelet agglutination mediated through the platelet receptor GPIb. Deletion of the vWF propeptide produced a dimeric vWF molecule that failed to mediate platelet agglutination, suggesting that multimerization is required for vWF to attain functional GPIb binding. This mature dimeric form of vWF, however, was fully capable of binding to and supporting stable secretion of factor VIII. A vWF mutant with an altered propeptide cleavage site formed large multimers of uncleaved pro-vWF that functioned in platelet agglutination. However, this noncleavage mutant neither bound to or supported stable accumulation of factor VIII. Analysis of the vWF propeptide, expressed independently, demonstrated that it could not bind factor VIII or stabilize its secretion. These results show that the dimeric mature vWF subunit is sufficient to bind and stabilize factor VIII and that the presence of uncleaved vWF propeptide inhibits both factor VIII binding and stabilization.     

A case of Osler-Rendu disease with simultaneous thrombopenia and a factor VIII inhibitor]

Long-term determinations of haemostasis factors in a case of Osler's telangiectasia revealed the temporarily simultaneous existance of periods of thrombocytopenia, a decrease of prothrombine and a reduction of the fibrinogen and plasminogen level. These findings considered as signs of consumption coagulopathy coincided with an increased bleeding tendency of the patient. The correlation existing between the clinical symptoms and the observed disorders of coagulation may possibly be explained by the appearance of an intravascular coagulation during the late period of the haemorrhagic diathesis, which could be proved by the simultaneous increase of fibrinogen degradation products. Moreover, the patient's plasma was capable of strongly inhibiting factor VIII of a normal plasma. The possible influence of plasmatic disorders of coagulation which are caused by secondary reasons on the clinical picture of a haemorrhagic diathesis primarily based on vascular conditions is discussed.     

Combined deficiency of factor V and factor VIII. A report of another case.

A patient with combined factor V and factor VIII deficiency is presented. The bleeding manifestations were: easy bruising, post-traumatic bleeding, bleeding after tooth extractions. The main laboratory feature was a prolonged partial thromboplastin time which was corrected by the addition of adsorbed normal plasma but not by the addition of normal serum, hemophilia A plasma of another patient with combined factor V and factor VIII deficiency. The thromboplastin generation test was clearly abnormal and was corrected by the addition of adsorbed normal plasma but not by addition of normal serum. Prothrombin consumption was also defective. Prothrombin time was slightly prolonged too, Thrombin time, platelet and vascular tests were within normal limits and there was no hyperfibrinolysis. Factor VIII was 8% of normal, whereas factor V was 14% of normal. Factor VIII associated antigen was normal. All other clotting factors were within normal limits. The parents of the propositus were consanguineous (first cousins) but had normal factor V and factor VIII activity and normal factor VIII antigen. The same was true for other family members. The hereditary transmission of the condition appears autosomal recessive.     

Fractionation of individual, biologically active factor VIII multimers

We have designed an electrophoretic system for the fractionation of individual, biologically active multimers of factor VIII. Human factor VIII, purified by gel filtration on Sepharose CL-2B from plasma cryoprecipitate, was submitted to electrophoresis without SDS on 2.0% polyacrylamide gels in 0.04 M Tris/0.06 M Tes buffer, pH 7.5. Staining with Coomassie blue revealed a series of protein bands. Measurement of electrophoretic mobility showed constant size intervals between adjacent bands. Electrophoresis in a second dimension, in the presence of SDS, resulted in an identical order of mobilities, suggesting that the different migration rates of factor VIII proteins in the first electrophoretic system were size- and not charge-dependent. After electrophoresis in the absence of SDS both factor VIII coagulant and ristocetin cofactor activities as well as factor VIII-related antigen were recovered by elution from gel slices. The distribution of activity peaks resembled that of Coomassie-stained factor VIII proteins found in control gels. We thus demonstrate that an electrophoretic fractionation of factor VIII multimers is possible even at neutral pH where factor VIII activities are retained.     

Some characteristics of aggregates of IgG and plasma proteins in heat-treated factor VIII concentrates

Eight batches of commercial heat-treated and one untreated factor VIII concentrate from 6 producers were analyzed for their content of IgG, IgG subclasses, IgG aggregates and the presence of other plasma proteins combined with the IgG as well as for anticomplement activity. Methods used were thin-layer gel filtration, immuno-gel filtration, spot immuno-precipitate assay in a double antibody version and an agarose plate haemolysis inhibition assay of complement fixation. The IgG content varied from 0.1-6.90 g/l. In all preparations IgG existed as monomers and aggregates. Associated with the IgG were also found, at significantly increased amounts compared to normal serum and intravenous immunoglobulin, one to four of the following plasma proteins; fibronectin, fibrinogen, von Willebrand factor antigen, Clq, albumin and IgA. Three batches from two producers had high anticomplementary activity, presumably caused by the IgG aggregates. Two of these deviated strikingly from normal human serum pools in percent distribution of IgG subclasses. It is hypothesized that these aggregates can induce side effects or cause immunological aberrations.     

The effect of plasma von Willebrand factor on the binding of human factor VIII to thrombin-activated human platelets

The binding of 35S-labeled recombinant human Factor VIII to activated human platelets was studied in the presence and absence of exogenous plasma von Willebrand factor. In the absence of added von Willebrand Factor, platelets bound 210 molecules of Factor VIII/platelet when the unbound Factor VIII concentration was 2.0 nM (Kd = 2.9 nM). As the von Willebrand factor concentration was increased, the number of Factor VIII molecules bound/platelet decreased to 10 molecules of Factor VIII bound/platelet at 24 micrograms/ml of added vWF. Addition of an anti-vWF monoclonal antibody that inhibits the vWF-Factor VIII interaction attenuated the ability of vWF to inhibit binding of Factor VIII to platelets. In contrast, addition of a control anti-vWF antibody that does not block the vWF-Factor VIII interaction did not affect the ability of vWF to inhibit Factor VIII binding to platelets. From the vWF concentration dependence of inhibition of Factor VIII-platelet binding, a dissociation constant for the Factor VIII-vWF interaction was calculated (Kd = 0.44 nM). To further elucidate the role that vWF may play in preventing the interaction of Factor VIII with platelets, the platelet binding properties of a Factor VIII deletion mutant (90-73) which lacks the primary vWF-binding site was studied. The binding of this mutant was unaffected by added exogenous vWF. These observations demonstrate that Factor VIII can interact with platelets in a manner independent of vWF but that excess vWF in plasma can effectively compete with platelets for the binding of Factor VIII. In addition, since cleavage of Factor VIII by thrombin separates a vWF-binding domain from Factor VIIIa, we propose that activation of Factor VIII by thrombin may elicit release of activated Factor VIII from vWF and thereby make it fully available for platelet binding.     

Platelet-derived microparticles express high affinity receptors for factor VIII.

Factor VIII is a cofactor in the tenase enzyme complex which assembles on the membrane of activated platelets. A critical step in tenase assembly is membrane binding of factor VIII. Platelet membrane factor VIII-binding sites were characterized by flow cytometry using either fluorescein maleimide-labeled recombinant factor VIII or a fluorescein-labeled monoclonal antibody against factor VIII. Following activation by thrombin, most platelets bound factor VIII within 90 s. In addition, over the course of several minutes, membranous vesicles (microparticles) were shed from the platelet plasma membrane and each microparticle bound as much factor VIII as a stimulated platelet. Over 30 min, stimulated platelets (but not microparticles) lost the capacity to bind factor VIII. Factor VIII bound saturably to microparticles from platelets stimulated with thrombin, thrombin plus collagen, or the complement proteins C5b-9. The binding of factor VIII was compared to factor V, a structurally homologous coagulation cofactor. Analysis of microparticle binding kinetics yielded similar on and off rates for factor VIII and factor Va and KD values of 2-10 nM. In the presence of 20 nM factor Va, the binding of factor VIII to microparticles was increased, and there was a comparable increase in platelet tenase activity. At higher factor Va concentrations, factor VIII binding and tenase activity were inhibited. Conversely, factor VIII had a similar dose-dependent effect on factor Va binding and platelet prothrombinase activity. Synthetic phospholipid vesicles containing phosphatidylserine competed with microparticles for binding of factor VIII and factor Va. These studies indicate that activated platelets express a transient increase in high affinity receptors for factor VIII, whereas platelet-derived microparticles express a sustained increase in receptors. The binding characteristics of platelet membrane receptors for factor VIII are similar to those for factor Va.     

Immunological Characterization of Purified Anti-Haemophilic Factor A (Factor VIII) which corrects Abnormal Platelet Retention in Von Willebrand's Disease

ABNORMAL platelet retention in a column of glass beads, which has been described in cases of Von Willebrand's disease (VWD)^1–6, is corrected in vitro by cryoprecipitate prepared from normal plasma and partially purified factor VIII concentrates^6–9 although fibrinogen is ineffective (unpublished results). This suggests that factor VIII is responsible for the correction. We now have further evidence to support this suggestion.     

Fibronectin and factor VIII-related antigen in acute leukaemia

The glycoprotein fibronectin is, as well as by various other cells, also produced in leucocytes and is said to play an important role in malignant transformation of cells. Therefore, the behaviour of plasma fibronectin and of factor VIII R:AG was investigated in acute leukaemia in order to prove their significance as prognostic and therapeutic markers (method: electroimmunoassay). In patients with acute myeloid leukaemia (n = 29) and acute lymphoblastic leukaemia (n = 11) no significant changes in fibronectin concentration could be evaluated. Fibronectin levels declined significantly only during therapy with asparaginase in patients with acute lymphoblastic leukaemia, probably as a result of disturbed synthesis in the liver. Using crossed immunoelectrophoresis against fibronectin antiserum, one normal and one slower migrating antigen (FN:C) could be observed in nearly all plasma samples in patients with acute leukaemia. By means of in vitro tests with highly purified substances and intermediate gel electrophoresis it could be shown that FN:C represents fibronectin which has bound fibrinogen, probably crosslinked by activated factor XIII. Factor VIII R:AG was found to be greatly raised in patients with acute leukaemia--up to 1400% of the normal level. Increased levels correlated well with a worsening of the disease. The protein seems to be suitable for estimating the activity and prognosis of acute leukaemia.