Doris Menache

Rapid methods for isolation of human plasma fibronectin

Simplified procedures have been developed for isolation of human plasma fibronectin by affinity chromatography on gelatin-agarose. In one method, fibronectin is eluted with 3 M urea, and this reagent is quickly removed by adsorbing the protein onto heparin-agarose, followed by 0.4 M NaCl elution. In a shorter process, fibronectin is eluted from gelatin-agarose simply by decreasing the buffer pH below 6. After lyophilization the purified protein can be readily dissolved in water. The fraction not adsorbed to gelatin can be used to purify other proteins, including factor VIII whose procoagulant activity is quantitatively recovered.

Pharmacokinetics of von Willebrand factor and factor VIIIC in patients with severe von Willebrand disease (type 3 VWD): estimation of the rate of factor VIIIC synthesis

Nine patients (10 infusions) with a confirmed diagnosis of type 3 VWD were infused with von Willebrand factor (human), a preparation of von Willebrand factor (VWF) with a very low factor VIII content. Each patient was infused with one dose of approximately 50 or 100 iu ristocetin cofactor activity (VWF:RiCoF) per kg body weight. Bleeding times were performed during the 24 h period after infusion. Plasma samples were obtained over the 96 h period after infusion and were analysed for factor VIII coagulant activity (FVIIIC), VWF:RiCoF, von Willebrand factor antigen (VWF:Ag), and multimers. The FVIIIC data were analysed by non‐linear least‐squares analysis assuming constant FVIIIC ‘synthesis’ and exponential decay. The VWF data were fitted for exponential decay. The average decay rates for FVIIIC, VWF:RiCoF and VWF:Ag were 0.041, 0.061 and 0.056 respectively. The average calculated ‘synthesis’ rate for FVIIIC was 6.4 u/dl/h. The synthesis of FVIIIC was slightly faster and the decay slightly slower following the infusion of 100 iu VWF:RiCoF/kg than of 50 iu VWF:RiCoF/kg. Correction of the bleeding time was strongly dose dependent. At 4 h post infusion the median bleeding time was 9 min following a dose of 50 iu VWF:RiCoF/kg versus 3 min with a dose of 100 iu VWF:RiCoF/kg. There was no decrease in the bleeding time until the level of VWF:Ag or VWF:RiCoF reached >100 u/dl.

The mechanism of activation of human prothrombin by an activator isolated from Dispholidus typus venom.

Purified human prothrombin was activated, both in the absence and in the presence of thrombin inhibitors (diisopropylfluorophosphate or hirudin), by a coagulant principle isolated from Dispholidus typus venom. The process of activation was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In the absence of thrombin inhibitor, prolonged incubation of prothrombin with the purified venom yielded thrombin, fragment 1 (F 1) and fragment 2 (F 2). In the presence of diisopropylfluorophosphate, which in the experimental conditions used inhibited only partially the thrombin generated activity, products obtained upon activation of prothrombin by venom were F 1 and a two-chain, disulfide-bridged protein of 58 000 daltons called meizothrombin (des F 1). In the presence of hirudin, which fully inhibited thrombin generated activity, prothrombin activation by the venom did not liberate any fragment, but prothrombin was converted to a derivative composed of two disulfide-bridged polypeptide chains of 48 000 and 37 000 daltons, called meizothrombin. These results are similar to those reported by others when studying the process of prothrombin activation by Echis carinatus venom and allow to conclude that Dispholidus typus venom cleaves a bond linking the A and B chains of thrombin, converting prothrombin into meizothrombin. This enzyme is then responsible for the cleavage of the bond linking F 1 and F 2 and the bond linking F2 the A chain of thrombin.

Congenital Fibrinogen Abnormalities

Congenital fibrinogen abnormalities are rare disorders and include afibrinogenemia, dysfibrinogenemia, and hypofibrinogenemia. Afibrinogenemia and dysfibrinogenemia are two relatively well-defined entities, the former being a quantitative defect characterized by the lack of circulating fibrinogen, whereas the latter is characterized by the presence of a qualitatively abnormal and functionally defective fibrinogen mdecule. Following the suggestion of Beck,’ a nomenclature similar to that formerly used for abnormal hemoglobins has been adopted and abnormal fibrinogens are designated according to the city of origin of the patient. Hypofibrinogenemia is less well defined. It may occur in both parents of a patient with afibrinogenemia where it clearly represents the heterozygous state. Low levels of fibrinogen determined by immunologic methods may also be found in some patients with an abnormal fibrinogen molecule (hypodysfibrinogenemia) , e.g., Bethesda 11,2 Bethesda 111,3 Chapel Hill,* Giessen 11,6 Leuven,6 New York,‘ Parma,8 Philadelphia and Valencia.lO However, it is still not clear whether some patients with hypofibrinogenemia represent a totally separate condition.

Human fibrinogen heterogeneities. Preparation and characterization of γ and γ′ chains☆

Abstract When normal human fibrinogen is subjected to DEAE-cellulose gradient elution chromatography, two major peaks (peak 1 and peak 2 fibrinogen, respectively) are observed. This behavior is attributable to the presence, in peak 2 molecules, of a γ chain variant termed γ′. A simple and efficient procedure is described for separating the S-carboxy-methyl derivatives of γ and γ′ chains by DEAE-cellulose gradient elution chromatography. Gel isoelectric focusing of γ or γ′ chain fractions revealed several bands. Those produced from the γ′ chain fraction, consistent with its known charge characteristics, were found in the more anodal γ chain regions; those from the γ chain fraction were displayed in more cathodal positions. Mutant γ chains (termed γParis I) from a congenitally abnormal fibrinogen molecule (fibrinogen Paris I) also expressed the γ/γ′ charge heterogeneity, suggesting that the difference between γ and γ′ chains is attributable to post-transcriptional chain modification.

Fetal fibrinogen and fibrinogen Paris I: Comparative fibrin monomers aggregation studies

Abstract Comparative studies have demonstrated that in human cord the conversion of fibrinogen to fibrin by thrombin led to the appearance of fibrin monomers that had an aggregation profile that differed from the adult. The aggregation rate and the final recorded absorbance were respectively slower and lower for the cord fibrin monomers. The action of thrombin on fibrinogen derived from bovine fetus led to the appearance of fibrin monomers that had the same characteristics described for human cord monomers. In addition two different features were observed: the monomers had an inhibitory effect on the aggregation of cow monomers and a second population “soluble fibrin monomers” was observed. Whether these differences were related to species specificity or to the degree of gestation is not known. The study of the congenital and inherited fibrinogen Paris I showed that the aggregation step of the fibrinogen to fibrin conversion was abnormal. In addition the fibrin monomers had an inhibitory effect on the aggregation of normal fibrin monomers and, a second population “soluble fibrin monomers” was observed. The fact that the same observations were made while studying the aggregation of bovine fetus fibrin monomers and the abnormal fibrinogen Paris I may be fortuitous. It may also allow to raise the question whether or not some cases of abnormal fibrinogen are related to the persistance of fetal fibrinogen. Biochemical data would still be necessary to sustain such a hypothesis.

The Use of Factor IX Concentrates for Patients with Conditions other than Factor IX Deficiency

The increasing availability of factor IX concentrates have extended their use in conditions other than factor IX deficiency and especially in combined acquired deficiencies of vitamin K dependent clotting factors.

Antithrombin III concentrates

The general characteristics of antithrombin III (AT III) concentrates available in the United States are described in this article. The effectiveness of AT III concentrates in the prevention and treatment of thrombotic episodes in patients with hereditary AT III deficiency are summarized, and the use of this product in various conditions with acquired AT III deficiency are reported.

Inactivation of HIV in antithrombin-III concentrate by pasteurization

osmotic lysis. We obtained results by this method that are the reverse of the (direct) urea lysis test, i.e., no lysis of Jk3 but lysis of Jk-3 red cells. These results are not consistent with the conclusion of Edwards-Moulds and Kasschau.3 in that impairment of urea transport in Jk:-3 red cells is clearly implicated. This latter method also has been adapted to a microplate method that we call the reversed urea lysis test (Table 1). This test has an advantage over the direct test in that the results do not change with extended incubation in the urea solution. Over a 2-year period we have tested a totalof 52.908 blood donor samples by both urea lysis methods without finding a Jk-3 blood. However, six different, known Jk-3 samples were tested blind during this period and gave the expected results. DOUGLAS C. J. MCDOUGALL, FIMLS, MSc MARTIN MCGREGOR. FIMLS Nor th-h t Thames Regwwl Transfuswn Centre Crescent Drive B r e n t w d Errex, CM15 8DR England

The purification of human acarboxy prothrombin. Characterization of its derivatives after thrombin cleavage

Abstract After administration of vitamin K antagonists, the liver is able to give rise to a prothrombin molecule which is different from normal prothrombin. This abnormal protein has been isolated using plasma derived from patients under either nicoumalone or ethylbiscoumacetate therapy, and designated human acarboxy prothrombin. After purification, the electrophoretic and immunological characteristics of this protein were studied before and after cleavage by human purified thrombin. The methods used were sodium dodecyl sulfate gel electrophoresis, disc gel electrophoresis at alkaline pH and Ouchterlony double diffusion technique. The results indicate that human acarboxy prothrombin has apparently the same molecular weight as normal prothrombin but has a slower electrophoretic mobility at alkaline pH. Analysis of the fragments obtained by thrombin cleavage shows that Prethrombin 1 derived from normal or acarboxy prothrombin have the same molecular weight and the same electric charge. Fragment 1 derived from acarboxy prothrombin has the same molecular weight as normal Fragment 1 but differs by its slower mobility at alkaline pH and by the lack of some antigenic determinants as demonstrated by the use of both an antiserum specific for normal prothrombin and an antiserum specific for normal Fragment 1.