Blood clotting in minimally altered whole blood

The sequences of events regulating thrombin generation during tissue factor-initiated clotting in whole blood at 37 degrees C in which the contact pathway was suppressed with corn trypsin inhibitor are studied using quantitative Western blotting of factor V, prothrombin, platelet factor 4, antithrombin III, and fibrinogen. In addition, fibrinopeptide A (FPA), thrombin-antithrombin III (TAT) complex formation, and prothrombin fragment 1.2 (F1.2) were measured via commercially available enzyme-linked immunosorbent assays (ELISAs). In a typical experiment initiated with 40 pmol/L recombinant tissue factor, visual clot time (4.5 minutes), was preceded by significant cleavage of factor V resulting in 65% factor Va heavy-chain generation but only 10% light- chain formation. At this point, 50% of the platelet factor 4 is released, suggesting that half (approximately 700 pmol/L) of the platelet prothrombinase sites available have been generated. At clot time, approximately 15 nmol/L thrombin B-chain is present; however, analyses of FPA release demonstrate that only 15% of the thrombin is acting on fibrinogen. This thrombin is produced by the action of 7 pmol/L prothrombinase. The maximum rate of thrombin production is reached well after clot time and is consistent with the presence of approximately 150 pmol/L prothrombinase (at about 7 minutes). These results suggest that factor Xa is the limiting factor for thrombin generation. After 60 minutes, 75% of the initial prothrombin (1.24 mumol/L) is consumed yielding 400 nmol/ L prethrombin 2 and 360 nmol/l thrombin (B-chain) products. The sum of these values (800 nmol/L) is similar to the (corrected) F1.2 concentration determined by ELISA. The incomplete cleavage of prothrombin indicates both the prothrombinase complex and the formation of prothrombinase are inhibited in the reaction. TAT complex measured by ELISA is almost equivalent to B-chain concentration, but sodium dodecyl sulfate stable thrombin-antithrombin III complexes are not observed until well after clot formation and are never equivalent to ELISA-TAT values. At the point of clot formation, 80% of the fibrinogen is depleted from the fluid phase, whereas only 35% to 45% of the FPA is released, suggesting a significant incorporation of uncleaved fibrinogen into the initial clot formed.     

Allogeneic blood transfusion-induced enhancement of tumor growth: two animal models showing amelioration by leukodepletion and passive transfer using spleen cells [see comments]

Allogeneic blood transfusions have been reported to induce immunomodulation in recipients of blood products. While the mechanism(s) of this immunomodulatory effect is unknown, it has been suggested that this effect of allogeneic blood transfusions could adversely affect patients with a malignant disorder. These concerns have been supported by a number of nonrandomized, mainly retrospective, clinical studies which indicate that allogeneic blood transfusions can adversely affect prognosis following the surgical treatment of oncology patients. Recently, we have shown that allogeneic blood transfusions enhance primary tumor growth and increase metastatic pulmonary nodule formation in inbred mice. The tumor growth-promoting activity of allogeneic blood transfusions was studied also using outbred rabbits. In this present study, we demonstrate that the tumor growth-promoting effect of allogeneic blood transfusions is mediated by donor leukocytes and that this effect can be abolished by their removal before transfusion. We show also that the allogeneic blood transfusion tumor growth-promoting effect can be passively transferred to naive animals (both mice and rabbits) using spleen cells from allogeneically transfused animals. In these experiments, numbers of metastatic pulmonary nodules were significantly increased in both mice and rabbits that had received spleen cells from allogeneically transfused animals compared with those that had received spleen cells from syngeneically transfused animals, or from animals that had been transfused with leukodepleted allogeneic blood.     

Purification and characterization of factor VIII 1,689-Cys: a nonfunctional cofactor occurring in a patient with severe hemophilia A

We have purified the factor VIII from a CRM+ Hemophilia A plasma (90 U/dL VIII:Ag but 0 U/dL VIII:C) and analyzed the protein before and after thrombin activation by Western blotting with monoclonal antibodies (MoAbs). Normal or patient citrated plasma was ultracentrifuged, cryo-ethanol-precipitated and chromatographed on Sepharose 6B. The void volume fractions were reduced and subjected to ion exchange chromatography yielding material of specific activity approximately 1,000 U/mg protein (VIII:C or VIII:Ag). Factor VIII purified in this way from normal plasma is fully activatable by thrombin with proteolytic fragmentation as previously described by F. Rotblat et al (Biochemistry 24: 4294, 1985). Factor VIII 1,689-Cys has the normal distribution of factor VIII light and heavy chains prior to thrombin activation. After exposure to thrombin the heavy chain polypeptides were fully proteolysed but the light chain was totally resistant to cleavage. This is consistent with the demonstration in the patient's leucocyte DNA of a C to T transition in codon 1,689 converting Arg to Cys at the light chain thrombin cleavage site as previously described by J. Gitschier et al (Blood 72:1022, 1988). Uncleaved light chain of Factor VIII 1,689-Cys is not released from von Willebrand factor (vWF) by thrombin, but this is not the sole cause of the functional defect since the protein purified free of vWF has no coagulant activity. We conclude that the functional defect in factor VIII 1,689-Cys is a consequence of failure to release the acidic peptide from the light chain upon thrombin activation.     

Serotype and adhesion of Pseudomonas aeruginosa isolated from contact lens wearers

The purpose of the present study was to correlate the serotypes of Pseudomonas aeruginosa to the bacterial adhesion to contact lenses and human corneal epithelial cells. Twenty‐three strains isolated from contact lens wearers were used for the study. The bacterial serotypes were examined with a P. aeruginosa antisera kit. The attachment of bacteria on contact lenses or human corneal epithelial cells was determined by counting the number of adhered bacteria after incubation of the bacteria with contact lenses or corneal epithelial cells. The 23 ocular isolates belonged to seven serotypes. Strains of serotypes I, G and E were the three dominant serogroups and were more adhesive to contact lenses compared with other groups of the bacteria. The bacterial serotypes and the clinical sequelae were not strongly related. These results indicate that the surface characteristics of bacterial serotypes are related to the bacterial adhesion to the surface, but the pathogenesis of the bacteria may result from multiple factors.     

羟考酮对左旋氧氟沙星在人体内药物动力学的影响

目的研究羟考酮对左旋氧氟沙星(LVFX)在人体内药物动力学的影响。方法8名健康志愿者,单用LVFX或合用羟考酮,用HPLC法测定血药浓度。结果单用LVFX500mg后的药物动力学参数分别为tmax(1.562±1.050)h,cmax(6.6419±0.15860)μg/ml,AUC(47.65±11.29)h*μg/ml,T1/2(β)(7.034±0.941)h;合用羟考酮时,LVFX的药物动力学参数分别为tmax(1.125±0.641)h,cmax(7.652±2.594)μg/ml,AUC(48.74±10.58)h*μg/ml,T1/2(β)(6.275±0.588)h。两者除T1/2ka和cmax外,无显著性差异。结论羟考酮对LVFX在人体内药物动力学参数无影响。     

Hepatic infarction with absolute ethanol

Ethanol was used to embolize the hepatic artery in 7 dogs, producing occlusion proportional to the dose employed. On follow-up angiography at 1 and 4 weeks, occlusion was unchanged or more pronounced, indicating that such embolization is permanent. Because ethanol is nonviscous and easy to use, it is effective in occluding the hepatic arterial system. Perisinusoidal fibrosis is produced.     

Subacute scrotal pain: evaluation of torsion versus epididymitis with MR imaging

Torsion knot and whirlpool patterns result from the twisting of the spermatic cord, which produces distortion of cord structures. These two findings, along with spermatic cord vascularity, were used to prospectively differentiate testicular torsion from epididymitis in 11 patients with subacute scrotal pain who underwent magnetic resonance (MR) imaging. The final diagnosis was established clinically in four patients and surgically in five. MR imaging enabled the recognition of each entity without error. Retrospective review of findings highlighted other potential discriminating features, the most important of which were testicular size and vascularity. Because of its ability to highlight vessels and detail normal and abnormal intrascrotal anatomy, MR imaging in this small series enabled the differentiation of subacute torsion from epididymitis with 100% accuracy. This high degree of accuracy warrants further investigation.