Biochemical properties of the fibrinogen component of a fibrin glue before and after severe dry heat treatment.

Functional biochemical properties of 5 batches of the fibrinogen component of a fibrin glue produced by the ZLB Central Laboratory, Bern, each consisting of 4 different in-process samples (taken after the first and second precipitation step, lyophilization, and dry-heat treatment) were studied in vitro. We focused our attention on the effect of the anti-viral treatment of the lyophilized product by dry heat for 1 h at 100 degrees C. A slight reduction in maximal turbidity of all heat-treated samples was observed during the clotting assay compared to nontreated samples. Treatment with dry heat did not result in generation of fibrinogen fragments that might accelerate tissue-plasminogen-activator (t-PA)-enhanced plasminogen to plasmin conversion. The time course of fibrin cross-linking by factor XIII showed no differences between heated and unheated samples. This result indicates that exposure of the fibrinogen component to severe heat neither reduced activity of factor XIIIa nor affected the correct alignment of cross-linking sites in polymerized fibrin. Incubation of fibrinogen with thrombin, plasminogen, and t-PA resulted in a slightly enhanced degradation of fibrin derived from the heat-treated samples. The amount of residual moisture, determined to be within the range of 0.6-2.1% before heat treatment, did not influence clotting, cross-linking, and fibrinolysis parameters. In conclusion, the virus inactivation treatment by dry heat for 1 h at 100 degrees C induces no significant alterations of the in vitro biochemical properties of the fibrinogen component of this fibrin glue.

New variant of type II von Willebrand's disease with structural abnormality of plasma von Willebrand factor in a patient with very mild bleeding history.

A new variant of von Willebrand's disease has been discovered in 2 members of a Macedonian family of 6. The proband, an 8-year-old boy, showed a prolonged bleeding episode on 1 occasion. Ristocetin-induced platelet aggregation and bleeding time were normal. In plasma, ristocetin cofactor activity (RCo) and von Willebrand factor (vWf) antigen were reduced to the same clearly low level. The determination of vWf antigen of platelets resulted in borderline values, while RCo was clearly reduced. Low- and intermediate-resolution agarose gel electrophoresis showed absence of the largest multimers in plasma vWf, and slight reduction in platelet vWf. High-resolution gels revealed abnormal multimeric structure only in plasma vWf. The smaller multimers could be resolved in a broad central band flanked by 4 fainter satellite bands; however, satellite bands close to the central band were more intense, and more distant ones were fainter, compared to normal plasma. The central band of the fastest-moving multimer was markedly intensified, and the mobility of the whole quintuplet was slightly reduced. Heredity seems to be autosomal-dominant. No mutation was found in exon 28 of the vWf gene. Because there was only 1 mild bleeding episode in the family, this structural variation seems to have only little clinical consequence. We conclude that this vWf abnormality is different from those observed in other type II variants previously described. Based on the revised classification by the International Society on Thrombosis and Haemostasis, we proposed designation type 2A-Bern for this new subtype.

Quantitative and qualitative analysis of platelet GPIb and von Willebrand factor in liver cirrhosis.

Numerous abnormalities of plasmatic coagulation and platelet function may contribute to the bleeding in liver cirrhosis with a defective platelet-von Willebrand factor interaction being a potential mechanism. To analyze GPIb and von Willebrand factor in cirrhosis, we quantified the number of GPIb molecules on the platelet surface by flow cytometry, assessed the total (and indirectly the internal) pool of GPIb by ELISA and measured the circulating amount of glycocalicin in plasma as a measure of proteolytic activity and platelet turnover. Von Willebrand factor was characterized by ELISA, by its ristocetin-cofactor activity and by multimer analysis. Botrocetin-induced agglutination was used for functional analysis. The data from 8 well-characterized cirrhosis patients indicate that total GPIb is insignificantly increased to 46,000 +/- 5,000 molecules/P (normal: 39,500 +/- 2,000 [SEM]), surface-GPIb is normal with some variability and that the glycocalicin levels are 2-3 times higher than would be expected from the platelet count (= 100 +/- 5 x 10(9)/l). Von Willebrand factor antigen levels and activity were 400-500% of normal with a 22% reduction of the high molecular weight multimers. A significant hyperagglutination response to botrocetin was observed with platelets from both patients and controls using patient plasma as a source of von Willebrand factor. In conclusion, a hyperresponsiveness rather than a defective platelet-von Willebrand factor interaction can be observed in cirrhosis which may compensate for other hemostatic problems and appears to be mediated primarily by increased levels of von Willebrand factor.

Triplet structure of von Willebrand factor reflects proteolytic degradation of high molecular weight multimers.

High molecular weight (HMW) and low molecular weight (LMW) forms of von Willebrand factor (vWF) were isolated from normal human plasma in the presence of protease inhibitors. HMW and LMW vWF preparations were subjected to reduction of interdimeric disulfide bridges under mild reducing conditions. Following sodium dodecyl sulfate electrophoresis in 3% agarose, the vWF bands were detected by immunoblotting with a polyclonal rabbit anti-vWF antiserum as well as with two monoclonal antibodies directed against epitopes located in the NH2-terminal (MAb 418) or in the COOH-terminal (MAb 9) region of the vWF subunit. Our results suggest that the slowest migrating band of the dimeric triplet set of LMW vWF represents an asymmetric structure composed of an intact subunit to which one NH2-terminal and one COOH-terminal fragment are linked by disulfide bridges. The intermediate band of the first triplet of LMW vWF strongly reacted with MAb 9 but not with MAb 418, indicating that it represents a dimer of COOH-terminal fragments. The fastest migrating band of the same triplet is apparently a dimer of the NH2-terminal fragments because it reacted with MAb 418 but not with MAb 9. Each next higher family of triplets seems to contain one more asymmetric fragment of dimeric size. These results are compatible with a model according to which LMW forms of vWF are derived from HMW vWF by proteolytic cleavage in the circulating blood.

Multimeric analysis of von Willebrand factor by vertical sodium dodecyl sulphate agarose gel electrophoresis, vacuum blotting technology and sensitive visualization by alkaline phosphatase anti-alkaline phosphatase complex.

To detect von Willebrand factor multimers in plasma samples and factor VIII concentrates, a vertical discontinuous SDS electrophoresis was developed. A vacuum blotting system allowed to improve the transfer to the nitrocellulose membrane. The visualization of the separated multimers was sensitized by applying an alkaline phosphatase anti-alkaline phosphatase staining technique. The reported method clearly shows structural abnormalities of von Willebrand factor and deficiency of high multimers, the vacuum transfer is efficient and the sensitivity of the staining system is very high.

Double-blind randomised clinical trial of pentoxiphyllin in vaso-occlusive sickle cell crisis.

Frequent painful sickle cell crisis leads to a high number of active days lost due to morbidity or mortality. Only one of the specific drugs has been shown to be efficacious in a controlled clinical trial in the USA. The efficacy and the appropriateness of a drug acting on the erythrocytic membrane, pentoxiphyllin, was investigated in a randomised, double blinded clinical trial in the treatment of acute incapacitating crisis. In the 36 patients studied in a rural hospital in Togo (West Africa) where sickle cell disease is frequent (0.51% of all births), the mean (standard deviation = SD) duration of inpatient treatment was significantly shorter in the actively treated group active drug: 77.6 (40.19) hours, placebo: 102.4 (25.31) hours, difference between means: 24.8 (95% confidence intervals (= 95% CI) 2.02-47.5) hours); p = 0.03. This marked effect of active treatment was evident during the first 48 hours only. The suitability of this therapeutic regime within the studied area is discussed.

Absence of diabetes in a rural West African population with a high carbohydrate/cassava diet.

1028 (99%) of the 1038 inhabitants of the West African village of Agbave and a random sample of 353 (12.4%) of the population of 2850 in Kati, another West African village, were screened for diabetes. Also recorded were their anthropometric data, dietary habits, possession of antibodies to malaria, and serum IgG concentrations. About 85% of the study population consumed cassava root at least once a day. The mean (SD) capillary random blood glucose concentration was 5.1 (1.1) mmol/l in men and 5.1 (0.6) in women. The mean (SD) body mass index was 20.2 (1.8) in men and 20.7 (2.3) in women. The mean blood glucose was similar whether cassava was consumed once daily, more than once daily, or less than once daily. None of the 1381 subjects examined had diabetes. This finding suggests that a high carbohydrate/cassava intake (84% of a mean daily supply of 1916 calories) combined with a low protein consumption (8% of caloric supply) does not cause diabetes. This does not support the World Health Organisation hypothesis that malnutrition-related diabetes exists, at least not in this West African rural population.

Isolation of fibronectin under mild conditions.

Fibronectin was isolated from human blood plasma by affinity chromatography on immobilized Physiogel, a plasma expander made by chemical degradation of gelatin. Binding of fibronectin to Physiogel is weaker than to gelatin; elution could therefore be performed under rather mild conditions: at pH 6.5 and 30 degrees C. Loading of the sample at low temperature increases the capacity of the affinity column.

Fatty acid patterns during plasma fractionation.

When albumin is prepared from human blood plasma by the cold ethanol method, nonesterified long chain fatty acids present in the plasma do not strictly copurify with albumin. About half of them are lost to the varioius globulin fractions precipitated in the first steps of the fractionation procedure. A different behavior is observed for the short chain fatty acid, caprylic acid. On the other hand, if fractionation of plasma proteins is done by ammonium sulfate precipitation, both long and short chain fatty acids remain with the albumin.