bluuebunny/crossref_metadata_2025 huggingface dataset:
ACTIVE SITE-SELECTIVE LABELING OF THROMBIN WITH FLUORESCENCE PROBES USING THI0ESTER DERIVATIVES OF PEPTIDE-CHL0R0METHYL KETONES
Authors: Bock Paul E
Published: August 2018

Active site-directed inactivation of a serine protease with a thioester derivative of a peptide-chloromethyl ketone followed by reaction of the unique thiol group generatedin the presence of hydroxylamine with a fluorophore-iodoacetamide hasbeen investigated as a new method for covalent incorporation of extrinsic fluorescence probes into the active sites of blood coagulation proteases. The specificity of labeling by this method was evaluated by quantitation of the reactions between human thrombin, acetylthioacetyl-D-Phe-Pro-ArgCH2Cl (ATA-FPRCK) and 5-iodoacetamidofluorescein(IAF).ATA-FPRCK was synthesized by reaction of FPRCK with succinimidylacetylthioacetate and purified by chromatography on SP-Sephadex and Sephadex G10. Titrations of the loss of thrombin chromogenic substrate activity with ATA-FPRCK were linear, with end points of 1.1-1.2 mol ATA-FPRCK added/mol active sites, consistent with a reaction stoichiometry of 1 and the ∽90% purity of the compound estimated by reverse-phase HPLC.Inactivation of thrombin wasquantitatively correlated with incorporation of the thioester, with a maximum of 1.04 mol/mol active sites.IAF labeling of ATA-FPR-thrombin inthe presence of 0.1M NH20H yielded a maximu of 0.96 mol IAF incorporated/mol active sites in a reaction accompanied by loss of the thiol group. Incorporation of ATA-FPRCK wasdependent on thefunctional thrombin active site, asdemonstrated by less than 4%thioester or IAF incorporation for the enzyme previously inactivated with FPRCK. I conclude that active site-selective fluorescence labelingcan be achieved by the method described here with the advantage of a wide choice in the properties of theprobe incorporated. In addition, a 2.3-fold difference in fluorescenceintensity was observed for 2,6-ANS derivatives of ATA-FPR-thrombin andATA-D-Phe-Phe-Arg-thrombin, indicating that the spectral properties ofenvironmentally sensitive fluorescence probes are influenced by the structure of the peptide inhibitor.

Supported in part by a grant from the American National Red Cross.
TRANSMISSION OF HUMAN IMMUNODEFICIENCY VIRUS INFECTION TO HOUSEHOLD CONTACTS OF PERSONS WITH CONGENITAL HEMATOLOGIC DISORDERS
Authors: Lusher J M , Aledort L M , Hiltgartner M , Mosley J , Operskalski E
Published: August 2018

The Transfusion Safety Study is collecting data concerning the transmission of transfusion-acquired infections from patients with congenital hematologic disorders to household members. Of 233 patients for whom information is presently available, 128 (55%) were anti-HIV-positive. The 128 positive patients lived in 123 households with 174 members; 16 contacts were positive by EIA and immunoblot.

These data provide further evidence of relatively high risk of HIV infection of sexual contacts. The three anti-HIV-positive children are all infants born to anti-HIV-positive wivesof infected hemophiliacs. Passively acquired antibody has not been excluded for two; the third was positive at ten months of age. Thus, vertical transmission may be a very important mechanism of perpetuating the HIV reservoir.
EVALUATION OF HIV ANTIGEN AND ANTIBODIES TO p24 AND gp41 IN HEMOPHILIACS
Authors: Laurian Y , Allain J P , Verroust F , Leuther M , Lambert T , Larrieu M J
Published: August 2018

Two hundred and seventeen hemophiliacs, known to be HIV seropositive for at least one year, were tested for HIV antigen (HIV Ag) and antibodies to p24 (p24 Ab) and gp41 (gp41 Ab) in 1986. HIV Ag was measured using a sandwich-type immunoassay (Abbott Laboratories); this assay predominantly detects core antigen, with a sensitivity of 30 to 50 pg/ml. Antibodies to p24 and gp41 were titrated with separate competitive immunoassays using recombinant antigens (Abbott Laboratories); reciprocal titers were calculated by comparison to a reference curve obtained by serial dilutions of a standard antibody containing sample. Forty-seven of these 217 patients (21.6 96) are HIV Ag positive, with an undetectable or low titer of p24 Ab in most of them. The rest (170) of the patients are HIV antigen negative ; some of them also have a low titer of p24 Ab. All the patients have a high titer of gp41 Ab. The year of seroconversion to HIV is known for 106 patients. Eighty seroconverted between 1983 and 1985 ; 14 of them (17.5%) are HIV Ag positive (3 had clinical symptoms related to HIV infection, but none developed AIDS); 66 are HIV Ag negative (16 developed clinical symptoms, but no AIDS was observed). Twenty six patients seroconverted between 1979 and 1982 ; 12 of them (46 %) are HIV Ag positive ; 14 patients are HIV Ag negative with low (7 cases) or high (7 cases) p24 Ab titers. The table summarizes the clinical data of these 26 patients.

In conclusion, HIV antigen and antibody titer to p24 may be of predictive value in the definition of lower or higher risk of developing clinical complications related to HIV infection.
ENDOTOXIN INDUCES DIC BY BOTH TISSUE FACTOR (TF) AND PLASMINOGEN ACTIVATOR-INHIBITOR (PA-l) SECRETION
Authors: Baldus B , Gehrmann G , Witt W , Donner P
Published: August 2018

DIC-induced organ failure represents a major pathomecha-nism in endotoxemia. To investigate which components of the haemostatic system may be disturbed during endotoxemia anaesthetized male Wistar rats were injected with E. coli endotoxin at dosages of 1 ng/kg to 100 pg/kg body mass. Citrated blood samples were investigated for fibrinolytic activity by a dilute blood clot-lysis time assay (DBC-LT), for PA-I activity by a new Tunctional assay using immobilized rt-PA on 96-well microtiter plates and for TF activity by measuring the rate of thrombin formation after stimulation with a 2000-fold diluted thrombo plastin reagent.

Endotoxin induced a decrease in fibrinolytic activity measured as a prolongation of the DBC-LT at dosages from 75 ng/kg to 100 μg/kg. This effect appeared 2 h after the treatment with endotoxin and persisted for<3 h. In parallel, an increase (up to 7-fold compared to baseline values) in PA-I activity could be measured 2 h after endotoxin application. When the plasma samples were clotted either by kaolin-phospholipid reagent, thrombin or reptilase in the resulting serum PA-I activity was more than 50-fold versus baseline. TF activity generated after stimulation with diluted thromboplastin reagent increased to about 5-fold compared to baseline values. This effect was already significant 1 h after endotoxin injection.

The results of this study indicate, that endotoxin induces a destruction of the balance of the haemostatic system by increasing coagulability via stimulation of TF activity and by decreasing fibrinolytic activity by secretion of PA-I. Furthermore the present data provide evidence for activation of a latent form of PA-I during the clotting process.
MINI PLASMINOGEN-LIKE MOLECULE IN SEPTIC PATIENTS
Authors: Kodrich L , Porterie P , Lago O , Bergonzelli G , Sassetti B , Sanchez avalos J C
Published: August 2018

We have previously described (Thromb.Res.44(6),1986) an altered relation Plasminogen (Pig)/α2~antiplasmin (APL) (Plg/APL<0.6) in the plasma of septic patients. A probable explanation of the mechanism whereby this alteration takes place would be the degradation of Pig to fragments of lower molecular weight due to the action of leukocyte elastase.In order to confirm this we studied 10 patients with sepsis, which did not have clinicalorlaboratory evidence of disseminated intravascular coagulation or septic shock, with positive blood cultures for bacterial germs .Elastase-α1proteinase inhibitor complexes were measured by an enzyme-linked immunosorbent assay (mean:510±181.9ug/l;normal range:86±28.5ug/l). Pig and APL functional activities were assayed by the amidolytic method; Pig: 40±8.9%; normal range: 100±20% .APL: 95±10.1% formal range 100+20%. Two different behaviors were observed in the plasma Pig of these patients with regard to their capacity to bind to Lysi-ne-Sepharose 4B.0n the basis of this observation the patients were divided into two group.Group A(4 patients) only presented Pig activity in fraction 1 (Pig without lysine binding sites : LBS). Group B (6 patients) presented Pig activity in fraction 1 and in fraction 2 (Pig with LBS).The normal controls presented Pig activity only in fraction 2. All the fractions which presented functional Pig activity also presented immunologic Pig activity and developed areas of lysis in heated fibrin plates after activation with urokinase

It seems tenable the hypothesis that the action of the leukocyte elastase is responsible for the degradation of Pig and this modification in the molecule would give rise to a greater depuration thus explaining the marked drop of the plasmatic levels seen in septic patients.
ABNORMAL FIBRINOGEN (FIBRINOGEN NAPLES) CHARACTERIZED BY DETECTIVE INTERACTION WITH THROMBIN AND PLASMIN IN TWO YOUNG SIBLINGS WITH ARTERIAL THROMBOSIS
Authors: Minno G Di , Cerbone A M , Cirillo F , Colucci M , Semararo N , Santo G Di , Mattioli P L , Mancini M , Quattrone A
Published: August 2018

Prolonged thrombin time (partially corrected by calcium chloride) and normal reptilase time were found in the plasma of two siblings with arterial thrombosis. Their purified fibrinogen showed similar abnormalities as well as impaired fibrino-peptide release in response to thrombin, delayed polymerization of pre-formed fibrin monomers and normal sialic content. Binding of radiolabelled thrombin by patient's fibrin was 30% of normal. Supernatants from patients' fibrin clots contained abnormal amounts of thrombin (not adsorbed by fibrin) and caused abnormal enhancement of platelet aggregation and ATP secretion from platelets exposed to sub-threshold concentrations of ADP or epinephrine. Hirudin suppressed the enhancing effect of the supernatant and substitution of γ-thrombin for α-thrombin led to normalization of platelet response. Studies on fibrinolysis showed that the abnormal fibrinogen from these patients as well as its naturally occurring derivative fibrin are highly resistant to lysis by plasmin. Thus our data support the concept that, in addition to the enhanced activation of platelets by residual free thrombin, thrombosis in these patients is the result of an impaired sensitivity of fibrinogen the lytic effect of plasmin.
FIBRINOGEN MILANO III, A NEW ABNORMAL HEREDITARY FIBRINOGEN VARIANT WITH A DEFECT IN THE Aα-CHAIN
Authors: Bögli C , Hofer A , Furlan M , Beck E A , Baudo F , Redaelli R
Published: August 2018

A new congential dysfibrinogenemia, denoted as Milano III, was found in a 13-year-old girl with recurrent thrombophlebitis. Plasma of the patient exhibited prolonged thrombin and reptilase times. Plasma fibrinogen concentration, determined by a functional assay, was less than 0.2 g/1 while the immunological method revealed normal fibrinogen levels. Fibrinopeptide release, induced by thrombin, was normal whereas polymerization of fibrin monomers was delayed. Under conventional conditions for normal fibrin aggregation, with and without added calcium, the final turbidity of abnormal fibrin was léss than 10 % of normal fibrin turbidity. The abnormal fibrinogen strongly inhibited clotting of normal fibrinogen. Isoelectric focusing showed an abnormal Aα-chain with greatly increased anodic mobility suggesting that the net electric charge of the Aα-chain is similar to that of the γ-chain. SDS-PAGE as well as reversed phase HPLC of mercap-tolyzed fibrinogen showed normal Bβ- and γ-chains whereas the Aα-chain was degraded. The susceptibility towards degradation appears to be related to the molecular defect in the variant Aα-chain, since normal Aα-chain was not affected under the same conditions. Intact size of Aα-chain was preserved during preparative chromatofocusing of reduced fibrinogen chains. Fibrinogen preparations from the proposita's mother and father contained approximately equal amounts of both normal and abnormal Aα-chain. We conclude that fibrinogen Milano III in the proposita is a new homozygous functionally abnormal variant with a structural defect in the Aochain which is not located in the amino terminus.
FIBRINOGENS KYOTO AND TOCHIGI, EACH WITH AN APPARENT ABNORMAL MOL. WT. γ CHAIN, ARE CHARACTERIZED BY REPLACEMENT OF γ ASN-308 BY LYS AND γ ARG-275 BY CYS, RESPECTIVELY
Authors: Yoshida N , Terukina S , Matsuda M , Moroi M , Okuma M , Aoki N
Published: August 2018

Congenital inherited abnormal fibrinogens (Fbgs) Kyoto and Tochigi showed prolonged thrombin- and reptilase-time, normal release of fibrinopeptides A and B, normal cross linking ability and impaired polymerization of the fibrin monomer.

Purified Fbg analyzed on SDS-PAGE under the reduced condition in the system of Laemmli contained 50 % of an apparent lower mol. wt. γ chain (γ Kyoto)(mol. wt.= 48,000 compared with 50,000 for the normal) in Fbg Kyoto and an apparent higher mol. wt. γ chain (γ Tochigi)(mol. wt.= 50,500) in Fbg Tochigi. Apparent mol. wt. differences were also detected in reduced and carboxymethyl ated Fbg, Fbg fragment D1, and D2, but not in D3. This suggested that the abnormality of γ chains in both Fbgs is in γ 303-356.

Amino acid sequence analysis was performed for CNBr- or lysylendopeptidase-digested peptides of the γ chain or D1 peptides after fractionation on HPLC. In Fbg Kyoto, γ Asn-308 was substituted by Lys, and a deletion of short peptides corresponding to the mol. wt. difference of 2,000 could not be detected. In Fbg Tochigi, γ Arg-275 was substituted by Cys, and no abnormality of amino acid sequence was found in γ 303-356.

These results suggest that some lesions or conformations containing γ 275 and γ 308 will directly or indirectly affect polymerization of fibrin monomers. Although the reason for apparent mol. wt. differences is not known yet, SDS-PAGE in the system of Laemmli will be useful for the analysis of abnormal Fbgs.

Fbg Kyoto could not be separated into two or three populations and may contain hetero-dimer molecules, but Fbg Tochigi had unclottable Fbg with predominant γ Tochigi and may contain abnormal homo-dimer molecules and normal molecules.
CONCENTRATED DDAVP: FURTHER IMPROVEMENT FOR THE TREATMENT OF MILD F.VIII DEFICIENCIES
Authors: Ghirardini A , DiPaolantonio T , Solinas S , Papacchini M , Cutrera C , Mariani G
Published: August 2018

We have evaluated the pharmacological efficacy of a concentrated DDAVP preparation (40 μg/mL) (herein referred to as C-DDAVP) administered subcutaneously (s.c.) in mild (n=23) and moderate (n=2) hemophilia A patients. A comparison between the response to s.c. C-DDAVP and the "dilute" DDAVP (4 μg/ml) (administered s.c. in 16 patients with mild (n=13) and moderate (n=3) hemophilia A and i.v. in 18 patients with mild (n=16) and moderate (n=2) was also carried out. In all instances Desmopressin was given at a dose of 0.3 μg/kg b.w. in absence of bleeding. The increase of F. VIII:C (expressed as post/pre ratio) after s.c. C-DDAVP was 2,55 at 30', 3,50 at 60'and 3,21 at 120'. The comparison among the three schedules of DDAVP administration showed that s.c. C-DDAVP elicited an increase of F. VIII :C at least as high as that induced by the dilute DDAVP with differences not statistically significant.

C-DDAVP was also administered s.c. in 4 patients with type I vWD (platelet normal subtype) with a normalization of bleeding time 60' after s.c. C-DDAVP, concomitant with a rise of activities related to F. VIII/vWF complex. Side effects were modest and transient. We can conclude that s.c. C-DDAVP is equally effective and safe in comparison with the dilute brand with the advantage of the minimal adminstration volume (<1 mL).
THE EFFECT OF DDAVP ON PLASMA AND PLATELET VON WILLEBRAND FACTOR MULTIMERIC PATTERN AND BLEEDING TIME IN PATIENTS WITH CONGENITAL PLATELET FUNCTION DEFECTS
Authors: Oksanen K , Kekomäki R , Lindbom A , Myllylä G , Rasi V , Vahtera E
Published: August 2018

Eleven patients with congenital platelet function defects and prolonged template bleeding time (BT) were studied. All patients had significant bleeding tendencies and were classified as having platelet release defects.

After initial blood sampling and BT measurement, tranexamic acid ( 10 mg/kg ) and DDAVP ( 0.3 ug/kg over 15 minutes ) were infused, followed by second blood sampling at, 30 minutes and BT measurement at 60 minutes.

Plasma vWF multimeric patterns were analyzed from 11 and platelet vWF multimeric patterns from 7 patients. vWF multimers were identified by electrophoresis of plasma or platelet lysates in agarose containing 0.1% SDS and visualized by immunoblotting using alkaline phosphatase-conjugated anti-antibody.

Bleeding time was normalized by DDAVP in 5 patients, partly corrected in 3, and unchanged in 3. Those 4 patients with longest BT ( more than 20 minutes ) either showed no response (3) or were only partially corrected (1).

Plasma F VIII:c, vWf:Ag, and F VIII:Rcof all increased similarly in responders, partial responders, or non-responders. The multimeric patterns of plasma vWF were initially normal in all patients and showed increases of the largest multimers in all three groups. Platelet vWF analysis also showed increase of larger multimers after DDAVP infusion; these changes usually parallelled those of plasma vWF. Increase of the large multimers was observed as well in responders and partial responders as in non-responders.

DDAVP was effective in correcting the prolonged bleeding time of several patients with platelet release defect. The analysis of plasma or platelet vWF multimeric pattern seemed not to predict this response.
UREMIC PLASMA, AFTER INFUSION OF DESMOPRESSIN, IMPROVES THE INTERACTION OF NORMAL PLATELETS WITH VESSEL SUBENDOTHELIUM
Authors: Castillo R , Escolar G , Monteagudo J , Cases A , Garrido M , Ordinas A
Published: August 2018

Desmopressin (DDAVP) shortened the bleeding time and increased the platelet retention on glass beads and the platelet interaction on subendothelium measured by the Baumgartner perfusion system in 11 uremic patients in whom the three tests were abnormal previous to the treatment (1).

Patients were chosen at random out of a group of 30 with prolonged bleeding time and decreased platelet retention on glass beads. All tests were performed on blood drawn before and one and six hours after a single dose of DDAVP (0.4 μg/kg body weight). Perfusion experiments were carried out at a shear rate of 800 sec-1.

Levels of VIII:C, VIIIR:Ag and RiCof were at the upper limit of normality before DDAVP and significantly increased one hour after treatment. The multimeric structure of vWF in pretreatment plasma was normal ; one hour after DDAVP larger multimers appeared.

After injection of DDAVP, the perfusion studies using reconstituted blood with uremic PPP in presence of isolated normal platelets and washed red cells, showed a statistically increased surface coverage and platelet aggregate formation on subendothelium (p < 0.05 respectively when compared to the pretreatment values). In the same perfusion assays, the in vitro addition to pretreatment plasmas of 1 u/ml of purified vWF with normal multimeric structure, or purified VIII:C and vWF (1 u/ml each) did not modify the decreased platelet interaction on subendothelium.

These results confirm the shortening of bleeding time by DDAVP in uremic patients and reveal an increase of platelet interaction on vessel subendothelium mediated by a factor present in PPP. Besides, they show that the effect of DDAVP in these patients is not due to the quantitative increase of the plasmatic vWF and FVIII.

(1) Blood, 68, 2:337-342, 1986.
THE EFFECTS OF VASOPRESSIN ON FIBRINOLYSIS AND FACTOR VIII ARE NOT MEDIATED THROUGH V2 RECEPTORS
Authors: Grant P J , Hampton K K , Wiles P G , Prentice C R M
Published: August 2018

Vasopressin (aVP) mediates its effects on smooth muscle through V1 receptors and on the kidney via pharmacologically distinct V2 receptors. Infusions of aVP and its long acting synthetic analogue DDAVP both produce increases in factor VIII and fibrinolytic activity in man. V1 receptors are known not to mediate this effect, however it has been suggested that the FVIII response might be mediated by V2 receptors as patients with nephrogenic diabetes insipidus are reported to have no FVIII response to DDAVP. It remains unclear whether this is a true phenomenon or reflects tachyphylaxis to the high vasopressin levels found in nephrogenic diabetes insipidus. The aim of this study was to investigate whether the pharmacological V2 receptor blocker lithium alters the effect of aVP infusions on FVIII and fibrinolysis in man. 4 control subjects and 6 patients taking long term lithium therapy (mean serum lithium 1.09 mmbl/l) were infused with 2.0 units aVP over 1 hour. Samples were collected for assay of aVP, euglobulin clot lysis time (ECLT) and FVIII coagulant activity (FVIIIC) before and at the end of infusion. In the control subjects median aVP rose from 0.5 to 83 pg/ml at the end of infusion. FVIIIC rose frcm 100 to 333% and plasminogen activator activity (PAA: 106 /ECLT) from 198 to 437 units. In the lithium treated group median aVP rose frcm 0.5 to 68 pg/ml at the end of infusion. FVIIIC rose from 100 to 263% and PAA from 102 to 453 units. There was a significant correlation between the plasma aVP and FVIIIC (r = 0.89 p < 0.005) and PAA (r = 0.92 p < 0.001) in the control group and the lithium treated group (FVIIIC r = 0.81 p < 0.002; PAA r = 0.69 p < 0.02). There was no significant difference between the rise in either FVIIIC or PAA in the lithium treated group compared with controls. These results do not support the hypothesis that the action of aVP on FVIII or fibrinolysis is mediated by V2 receptors. The effects of aVP on haemostasis may either be mediated directly through a third class of receptor or indirectly by the release of an intermediate hormone.
NORMAL EXPRESSION OF THROMBOSPONDIN RECEPTOR SITES ON PLATELETS OF THREE GLANZMANN THROMBASTHENIC PATIENTS
Authors: McGregor J L , Boukerche H
Published: August 2018

A well characterised anti-thrombospondin (TSP) monoclonal antibody (Mab) LYP8 was used to investigate the presence of TSP receptors on Glanzmann thrombasthenic (G.T.) platelets. LYP8 inhibited platelet aggregation induced by low concentrations of thrombin (0.05 U/ml) or collagen (0.5 ug/ml). The presence of LYP8 did not affect the number of sites and Kd of 125I-fibrinogen binding to thrombin-stimulated normal platelets. Binding of LYP8 to normal platelets was minimal in whole blood (300 IgG molecules/ olatelet), increased in citrated PRP (1187 ± 209 IgG molecules/ platelet) and washed platelets (2967 ± 1278 IgG molecules/platelet) . Thrombin stimulation of platelets, washed in the presence of 2 mM calcium, increased the number of LYP8 binding sites (14917 ± 42n IgG molecules/platelet). Addition of EDTA (5mM) to thrombin-stimulated platelets did not reduce the number of LYP8 binding sites. The number of LYP8 binding sites on thrombin-stimulated platelets of three Glanzmann thrombasthenic patients (showing an absence of the glycoprotein (GP) lib and IIIa)was similar to normals in the presence of 2 mM calcium or 5 mM EDTA. In competitive binding, Mab LYP18 directed against the GPIIb-IIIa complex did not inhibit the binding of labelled monoclonal antibody LYP8. These results strongly suggest that TSP binds to a membrane receptor different from the GPIIb-IIIa complex in the presence of calcium or EDTA. This unidentified receptor may be GPIV also known as GPIIIb (Asch, A. et al. Clin. Res. 1986, 34:450A).
IMMUNOLOGICAL STUDY OF GLYCOPROTEINS AND ANTIGENS IN GLANZMANN'S THROMBASTHENIA HETEROZYGOTES
Authors: Wautier J L , Boi zard B , Gruel Y , Caen J P
Published: August 2018

To further explore the genetic transmission of Glanzmann thrombasthenia (GT) we have conducted a study of platelet glycoproteins and antigens using mono or polyclonal antibodies and cytofluorography. Twenty two members of 4 different families in which at least one member was affected with GT were explored. The reactivity with AP2 (anti GPIIbllla complex), AP3 (anti GPIIIa) antibodies, anti PLA1 and anti Leka was lower than 5% in the platelets of. the 5 patients with GT type I. The 9 obligatory carriers for GT trait were tested with the same technique. The mean value of reactivity expressed as per cent of normal with AP2 and AP3 were 59±11 and 60±12% respectively. The frequency of PLA1 was 0/9 and Leka_ M/9. When both antigens were expressed the amount detectable by cytofluorography was reduced (45-66%). The incidence of Leka_ appeared to be significantly higher from that of a normal population (6.8%). Since PLA1 is located on GPIIIa and Leka on GPIIb the difference on the expression of the two antigens could be secondary to a difference in the ratio GPIIb/GPIIIa in heterozygotes. This could be explained either by an allelic interaction or a linkage between the genes which code for GPIIb and Leka abnormally expressed in GT.
GLANZMANN'S THROMBASTHENIA: HEMOSTATIC EFFECTIVENESS OF PLASMA CRYOPRECIPITATE TRANSFUSION
Authors: Nichols W L , Habermann T M , Kaese S E , Bowie E J W
Published: August 2018

A 39-year-old 75 kg man with Type I Glanzmann's thrombasthenia developed recurring severe therapy-refractory nosebleeds, associated with nasal septal deformity, synechial scarring, and previous radiotherapy. During a 21 month period he required 16 hospitalizations for treatment of severe epistaxis. At initial hospitalization it was observed that his epistaxis ceased shortly after transfusion of 10 bags (units) of plasma cryoprecipitate (cryo), although transfusion with HLA-matched apheresis platelets from 3 donors, and topical therapy (cautery, packing), had been ineffective. Serum anti-platelet antibodies were not detectable by indirect immunofluorescence. During 9 subsequent hospitalizations his epistaxis stopped promptly (usually within 1 hour) following cryo transfusion (10 or 20 bags), including 6 occasions when cryo was the only therapy. On 6 additional occasions his epistaxis did not stop following cryo transfusion, but did stop after subsequent platelet transfusions or topical therapy. Eventually he underwent nasal septal reconstructive surgery surgery, and severe epistaxis has not recurred. Hemostatic studies, before and after cryo transfusions on 5 occasions, did not show improvement of platelet aggregation defects nor of Ivy bleeding times, although occasionally the volume of blood emanating from bleeding time punctures appeared decreased following cryo transfusion. Platelet glycoprotein (GP) IIb/IIIa antigen was measured in aliquots of 10 of the pools of cryo received by the patient (representing 100 bags of total volume 2200 ml), using an immunoradiometric assay (Nichols et al, Blood 68:300a, 1986). On average, the transfused cryo pools contained GP IIb/IIIa equivalent to 1.1 x 108 platelets/ml (range 0.6-1.7 x 108/ml. Our recently reported studies of blood bank cryo documented similar GP IIb/IIIa levels, and revealed that >93% of GP IIb/IIIa in cryo is present in the form of sedimentable membranous platelet macroparticles and microparticles. We hypothesize that the GPIIb/IIla-bearing platelet particles in transfused cryo might account for the improvement in hemostasis we frequently observed. We conclude that cryo transfusion deserves further study as a potentially useful therapeutic adjunct in promoting hemostasis in individuals with Glanzmann's thrombasthenia.
ABNORMALITIES OF CYTOPLASMIC [Ca++] IN PLATELETS FROM UREMIC PATIENTS STUDIED WITH AEQU0RIN AND INDO-1
Authors: Ware J A , Clark B A , Smith M , Salzman E W
Published: August 2018

Uremic patients have a hemorrhagic tendency, often with prolonged bleeding times and abnormalities of platelet function in vitro. Whether these defects result from plasma factors, abnormalities in platelet surface receptors, or intracellular mediators is unknown. Accordingly, blood was obtained from 16 patients with severe uremia (BUN >90), and platelets were washed, loaded with aequorin or indo-1, gel-filtered, and resuspended in either plasma or buffer. Of the 16 patients, 4 had template bleeding times greater than 12 minutes, but platelet aggregation in plasma was not consistently impaired. However, the rise in cytoplasmic [Ca++] in response to the Ca++-ionophore A23187 or ADP in aequorin-loaded platelets from the 4 patients with long bleeding times was much lower than in uremic patients with normal bleejljLng times or in normal volunteers. The reduced [Ca++] response was associated with decreased aggregation of gel-filtered platelets in buffer. Prolonged bleeding time was less consistently correlated with decreased responses to epinephrine or arachidonate. Suspending washed aequorin-loaded uremic platelets in normal plasma for 10-20 min did not reverse the decreased agonist-induced rise in [Ca++]; platelets from a normal donor resuspended in uremic pla^iya responded normally. The agonist-induced rise in [Ca++] shown by indo-1 was not abnormal in patients with prolonged bleeding times; however, uremic patients generally had higher indo-l-indicated basal platelet cytoplasmic [Ca++] than normal. We conclude that the hemorrhagic tendency in some patients with uremia (|s associated with abnormal intracellular platelet [Ca++] regulation marked by elevated resting [Ca++] and a decreased rise in cytoplasmic [Ca++] in response to certain agonists; this latter abnormality appears to be correlated with prolonged bleeding times.
HUMAN RECOMBINANT ERYTHROPOIETIN (rHuEpo) CORRECTS ANEMIA AND SHORTENS THE BLEEDING TIME (BT) IN UREMIC PATIENTS
Authors: Moia M , Casati S , Della Valle P , Passerini P , Ponticelli C , Mannucci P M
Published: August 2018

A hemorrhagic tendency and anemia are two major complications of uremia. There is a significant negative correlation between BT and hematocrit (Ht) in uremic patients. We studied the efficacy of rHuEpo in correcting both these abnormalities in 10 patients on chronic hemodialysis with severe anemia (basal Hb levels 6.2±0.8 g/dL). Seven patients had BT longer than 10 min., 5 had BT longer than 30 min. rHuEpo was given to the patients 3 times a week at increasing doses, from 24 U/Kg to a maximum of 432 U/Kg, aiming to reach Hb levels ⪖12 g/dL. BT was measured (with the template-like disposable device Simplate II) before starting rHuEpo therapy and after reaching the target Hb values. One patient (n° 6) was excluded from the study after 5 weeks of rHuEpo treatment because of development of an arterio-venous fistula thrombosis. The data are shown in the table.

Eight of the 9 patients who concluded the study had final BT shorter than 10 min. This demonstrates the efficacy of rHuEpo in correcting both anemia and prolonged BT in uremic patients.
ANALYSIS OF PATIENTS WITH A PROLONGED BLEEDING TIME
Authors: Nieuwenhuis H K , Zwaginga J J , Sixma J J
Published: August 2018

The bleeding time (BT) is one of the most important screening tests in patients with a bleeding tendency. Investigations of Von Willebrand Factor and aggregation tests form routine diagnostic tools to analyse a prolonged BT. In many patients, however, this approach fails to explain the bleeding tendency because no abnormality is found. In order to deal with this problem , we chose three methods: I) determination of platelet nucleotides and serotonin in all patients with a prolonged BT, independent of the results of aggregation tests, II) measurement of platelet adherence to purified collagen and the matrix of endothelial cells in a perfusion system, III) measurement of adherence of normal blood to matrix of patients' fibroblasts. I) In a group of 145 patients with a prolonged BT and a normal platelet count the diagnosis von Willebrand1's disease was made in 52 patients (36%), congenital Storage Pool Disease (SPD) in 27 pts (18%), defect of thromboxane synthesis in 4 pts (3%) and platelet function disorders with miscellaneous aggregation abnormalities in 23 pts (16%). No abnormalities were found in 39 pts (27%). Analysis of aggregation tests, disclosed normal aggregation tracings in many patients with SPD: 23% of 106 patients with congenital or acquired SPD had normal aggregation tests.

Adhesion studies were performed with the blood of 7 patients with an unexplained prolonged bleeding time. Two patients had a severe defect and one patient a mild defect of adherence. Further studies revealed the presence of an autoantibody on the platelets in one of these patients.

In order to diagnose vessel wall defects which may cause a prolonged BT, we studied the adherence of normal blood to the matrix of the fibroblasts of one patient. We found a decreased adherence, which suggests the presence of a vessel wall defect causing a prolonged BT.

We conclude that in many patients with a prolonged BT and normal VWF parameters and normal aggregation tests, SPD or adhesion defects may be responsible for a bleeding tendency.
SYNTHETIC PEPTIDE PROBES OF FACTOR VIII IMMUNOLOGY AND FUNCTION
Authors: Fulcher C A , Houghten R A , de Graaf Mahoney S , Roberts J R , Zimmerman T S
Published: August 2018

In order to develop specific immunologic reagents for mapping functionally important sites on FVIII, we have prepared rabbit polyclonal antibodies against synthetic peptides of FVIII derived from regions along the entire FVIII amino acid sequence. To date, a total of 70 peptides have been synthesized and characterized by amino acid and HPLC analysis. The peptides were coupled to keyhole limpet hemocyanin with glutaraldehyde as a linkage reagent and used to immunize rabbits. Antisera were tested by ELISA assay on polystyrene microtiter plates coated with either the peptide immunogen, or purified FVIII. The antisera were also tested for their ability to inhibit FVIII clotting activity and to react with separated FVIII polypeptides on immunoblots.Of the 70 peptides, all reacted with the peptide immunogen, 45 reacted with purified FVIII and 33 reacted with FVIII on immunoblots. Because we had obtained evidence that cleavage of the amino terminal region of the 80 kDa polypeptide may play a role in FVIII activation by thrombin, a series of partially overlapping peptides, 15 residues in length, were synthesized in this area. After affinity purifying these antibodies on columns of FVIII immobilized on agarose, adjusting the antibodies to equal antigen binding titers by dot immunoblotting and testing for inhibition of FVIII activity, only one antibody could strongly inhibit FVIII clotting activity. This inhibition could be blocked by the peptide itself at nanomolar concentrations and no significant inhibition could be shown by antibodies to partially overlapping peptides individually, or in combination. These data suggest that a site important to FVIII function can be localized to a 15 amino acid residue region of the 80 kDa polypeptide of FVIII. In addition, a second inhibitoryantibody was identified which was produced against a peptide in the carboxy terminal region of the 54 kDa thrombin fragment of FVIII and this area is currently being studied in a similar manner. In addition, two monoclonal anti-FVIII synthetic peptide antibodies have been produced which react with purified FVIII on immunoblots. One of these antibodies also functions as an immunoadsorbent when linked to agarose and FVII can be purified in this manner, using the synthetic peptide as eluant. It is evident that antibodies to synthetic peptides of FVIII can be useful probes of FVIII structure, function and interactions as well as being of use in FVIII purification.
INACTIVATION OF FACTOR VIII BY ACTIVATED PROTEIN C AND PROTEIN S
Authors: Fay P J , Chavin S I , Walker F J
Published: August 2018

Human factor VIII has been isolated from factor VIII concentrates. The isolated protein is composed of a heavy chain and light chain. The heavy chain was heterogenous with respect to molecular weight ranging from 110-170 kDa. The light chain appeared as a 81/84 kDa dimer, 'when factor VIII was treated with activated protein C in the presence of calcium and phospholipids factor VIII procoagulant activity was rapidly lost. Analysis of the activated protein C catalyzed cleavage products of factor VIII indicated that loss of activity was correlated with cleavage of the heavy chains. The heavy chains appeared to be converted into 93 kDa and 53 kDa peptides. A separate factor VIII preparation has been prepared that contained only a 93 kDa heavy chain as well as the 81/83 kDa light chain. When this preparation was inactivated with activated protein C, a pathway in which the 93 kDa peptide was degraded into a 68 kDa peptide which was subsequently degraded into 48 and 23 kDa polypeptides. This result suggested that the 53 kDa polypeptide was not derived from the 93 kDa domain of the heavy chain, but must have been derived from the variable molecular weight portion of the heavy chain. These results suggest that activated protein C catalyzed a minimum of four cleavages in the heavy chain. Activated protein C did not appear to alter the factor VIII light chain. Protein S has been observed to be a protein cofactor both the anticoagulant and proteolytic action of activated protein C with factor Va. It is thought that protein S forms a lipid bound complex with activated protein C which then can rapidly inactivate factor Va. When factor VIII was inactivated in the presence of both activated protein C and protein S the rate of activity loss was enhanced. The effect of protein S could be observed on the cleavage of the heavy chains and on secondary cleavages of the smaller products including the 93, 68, and 53 kDa polypeptides. In an analogous reaction, the addition of factor Xa has been observed to inhibit the inactivation of factor Va by activated protein C. The addition of factor IX to the factor Vlll-activated protein C reaction mixture resulted in the inhibition of factor VIII inactivation. The effect of factor IX was dose dependent. Finally, as both factor Va and factor VIII have structural similarities and are substrates for activated protein C the possibility that they might compete as substrates was tested. Factor VIII was observed to compete with factor Va for activated protein C. The concentration dependence of factor VIII inhibition of factor Va inactivation suggested that factor VIII and factor Va were equivalent substrates for activated protein C.
THE DIRECT DEPLETION AND RECOVERY OF HUNAN COAGULATION FACTOR IX FROM PLASMA USING IMMOBILISED MONOCLONAL ANTIBODY
Authors: Bessos H , Powse C V , James E
Published: August 2018

An immobilised monoclonal antibody (ESN1) was repeatedly used for the preparation of artificial factor IX (FIX) deficient plasma (AND), and subsequent recovery of bound FIX. Column depletion removed >99% of the FIX from plasma, without affecting other clotting factors. Results of FIX assays performed with AND showed excellent correlation (r=0.981) with those using congenital FIX deficient plasma for plasma obtained from normal subjects, from patients on warfarin treatment, and congenitally FIX deficient subjects. Moreover, 70-90% of bound FIX was each time recovered from the ESN1 column in active form by elution with 7 M urea. After concentration and absorbtion of the eluate with protein A Sepharose CL4B, western blotting of non-reduced^ and reduced FIX showed a single major band which co-migrated with conventionally purified FIX. Repeated use of the ESN1 column (>9 times) has not hindered AND preparation or the recovery of bound FIX in active form.
STUDIES ON PLATELET AGGREGATION BY IMPEDANCE AGGREGOMETRY AND ATP SECRETION IN NON-ANTICOAGULANT BLOOD
Authors: Heptner W , Suárez J R , Lütgendorf V
Published: August 2018

Investigations in vitro on the time-dependent increase in thrombin activityand platelet function have been used tocharacterize the kinetics of the clotting process in nonanticoagulated blood. The test procedures described involve great effort and expense and therefore are not suitable for routine tests in pharmacology and clinical pharmacology. The present contribution describes the determination of clotting times in ATP secretion in the Chrono-Log Whole Blood Aggregometer.

Blood was taken from healthy donors who had not used any drug in the two weeks before the trial. 0.5 ml blood wereimmediately transfered into siliconizedglass cuvettes containing 0.4 ml salineand 0.1 ml luciferin-luciferase cocktail prewarmed to 37°C. Impedance and luminescence were recorded continuously. Clotting at the electrodes is indicated by an immediate steep rise in both impedance and luminescence. Clotting time is defined as the time from diluting the blood in the cuvettes until the point at which marked elevation of these variables begins.

In the blood of twelve subjects the mean clotting time was 3.8 min and intersubject variation (SD) was 0.45 min. Drastic interindividual differences in response to collagen and ADP in citratedwhole blood were observed in the study group.

In vitro addition of 20 μl Fibraccel(Behringwerke AG, Marburg, FRG),a platelet factor 3 containing plateletextract decreased clotting time by 35 %(n=10). In the presence of 0.2 U heparin a slow and lona-lasting increase in impedance was seen. 1 g oral AspirinR didnot influence clotting time measured ex vivo.

The results indicate that whole blood aggregometry is a simple, fast, and precise method of determining blood clotting and the effects of drugs in a medium reflecting almost physiological conditions.
IMPORTANCE OF SOME MOLECULAR MARKERS OF HEMOSTATIC ACTIVATION FOR THE DIAGNOSIS, THE CHOICE AND THE CONTROL OF PREVENTIVE AND CURATIVE THERAPY OF D.V.T. AND P.P.S
Authors: Moriau M , Col-De Beys Ch , Golinska B , Lavenne-Pardonge E
Published: August 2018

The measurement, before and after treatment, of some molecular markers of hemostatic activation (βTG, PF4, TXB2, FPA, 6 keto PGFlα) and the determination of their increased coefficient ratio (Δ+/βTG/Δ+Δ FPA, 4+βTG/4+ PF4,4+ 6 keto PGF1α/Δ+ TXB2 combined with the estimation of other parameters (F.VIII:Ċ/F.VIIIR:AG), AT III, PROT. C, ELT with and without ischemia) permlted us to subdivide the deep venous thromboses in two groups

The first one or "simple D.V.T." was observed in normal subjects with or without varicoses, and without other main pathology and was consecutive to a simple plasma factors activation, the second one or "complicated D.V.T." appeared in patients with other pathology (infections, neoplasms, trauma, metabolic disorders) and was consecutive to a combined activation of plasma factors and platelets

A:before treat.,B:after anticoagul.,C:after anticoagul. + platelet inhibitors / * extreme values (MV ±2ŊIf anticoagulants (Heparine ancjfor VKA) alone were effective and sufficient for the treatment of "simple DVT" they were little or non active in the "complicated" forms and must be combined with platelet inhibitors to give the same result. The prevention of DVT in venous insuffisancy, consecutive or not to DVT, must be performed in the same way. A comparative study with various types of platelet inhibitors permited us to establish a scale of activity and to observe a benefic synergism with some combinations
BINDING OF NATIVE PLASMINOGEN TO FIBRIN AND TO SOME FI BRINOGEN/FIBRIN DERIVATIVES
Authors: Zamarrón C
Published: August 2018

In the fibrinolytic process: (a) fibrin provides a surface on which the major reactions of fibrinolysis occurs: the conversion of plasminogen to plasmin, the cleavage of fibrin by plasmin and the inhibition of plasmin by α2-antiplasmin, (b) some fibrinogen derivatives (e.g. the cyanogen bromide digested fibrinogen fragment denominated FCB-2) can exert stimulatory properties on the plasminogen activation and (c) the initial cleavage of fibrin by plasmin increases the rate conversion of plasminogen to plasmin.

The purpose of the present work has been to correlate these three aspects of the fibrinolytic process with the binding of native plasminogen (Glu-Pg) to fibrin (Fn) , fibrinogen (Fg) and Fn/Fg derivatives.

The Glu-Pg-Fg interaction, if exists, it is not detectable in equilibrium conditions by analytical centrifu gation. By using a solid-phase fibrin clot system (purified system) the Glu-Pg-Fn interaction gives the following dissociation constants: Kd=3.5×10−6 M and 1.2×10−5 m (unwashed and washed clots respectively). Being two the number of plasminogen binding sites per fibrin fibrin monomer. By activation with streptokinase or urokinase the amount of Pg required for an effective lysis of the fibrin clots is lower when the Pg is endogenous (inside the clot) versus exogenous (outside the clot).

The binding of the isolated fragments of the cyanogen bromide digested fibrinogen to Glu-Pg was studied by affinity chromatography on Glu-Pg-Sepharose. The only fragment bound to Glu-Pg and eluted with 10 mM ε-amino caproic acid (ε;-ACA) was the fragment denominated FCB-2 The soluble fibrin monomer after 20 min plasmin digestion also binds to immobilized Glu-Pg and it is eluted with ε-ACA.

Therefore, the binding of native plasminogen to fibrin and to some fibrinogen/fibrin derivatives is a determinant factor in the three aspects of the fibrinolytic process mencioned above.
ENDOGENOUS CIRCADIAN RHYTHM OF FIBRINOLYTIC PARAMETERS
Authors: Köhler M , Mivashita C , Friedl A , Littbarski S , Heiden M , Wenzel E
Published: August 2018

Recently, the fibrinolytic system was shown to be dependent on complex interactions of several proteins for which specific test systems have been developed. We report on the results in placebo groups of two drug trials which strongly suggest an endogenous circadian rhythm of these parameters. In the first trial, 6 male subjects consecutively received either different heparins or placebo (group A). In a double blind placebo controlled trial, 10 male subjects received either DDAVP or placebo (group B). Blood was obtained before (7:30 a.m.), and 1, 2, 3, 4, 6, 9, 12 h and, only in group A, 15 and 24 h after the injection (8:00 a.m.). Euglobulin precipitate (EP) was prepared immediately after centrifugation. Fibrinolytic activity (FA) and t-PA activity were measured according to Astrup and Miillertz (1952) and Verheijen et al.(l982), respectively. t-PA antigen was measured using a kit from Biopool, Umea. t-PA-inhibitor (INH) was determined using a commercial kit from Kabivitrum and the method described by Chmielewska et al.(l983). Statistical analysis was performed using ANOVA (t-PA antigen) and Kruskal-Wal1is test.

Results: In group A, t-PA antigen was significantly higher in the morning than in the evening. In contrast, t-PA activity and FA were low in the morning and gradually increased and reached maxima at 8 p.m. and 11 p.m., respectively. In group B, the same pattern was observed and all diurnal variations were statistically significant. Higher levels of t-PA-INH and t-PA-antigen were observed in the morning, and lower levels of t-PA and fibrinolytic activity. A reversed pattern was seen in the evening ( 8 p.m.). The two different methods for t-PA measurements showed the same variation^ although a c.lO-fold higher level was determined using the Kabivitrum test kit.
MONOCLONAL PURIFIED FACTOR VIII:C (MONOCLATE) TREATMENT IN A PREVIOUSLY UNTREATED HAEMOPHILIA
Authors: Parapia L , Minford A , Hamilton J B
Published: August 2018

Monoclate is a new generation of Factor VIII concentrate produced by purification using mouse monoclonal anti-Factor VIII:R antibody. As the- Factor VIII:C does not interact with the antibody it can be eluted by disrupting the Factor VIII:C - Factor VIII:R complex using a high concentration of calcium ions. The eluted Factor VIII:C is concentrated and purified. The method of manufacture has demonstrated efficacy in the elimination of infectious viral particles.

The first “virgin” haemophiliac to be treated by this has completed 20 weeks follow-up. The patient, a child of 18 months with a Factor VIII:C level of 2.8%, was treated with 190 × 4 units of the Factor VIII concentrate for a severe cut of the lower lip.

The HIV status has remained negative. The AST and ALT enzymes have remained within normal limits. Other parameters which have remained normal are Gamma GT, WBC and lymphocyte counts, T cell subsets and B cell ratios.

The patient has remained well and no side effects have been noted. Mouse antibody titres are being carried out and the results will be presented at the conference.