bluuebunny/crossref_metadata_2025 huggingface dataset:
INFUSION OF MONOCLONAL ANTIBODY IMMUNOAFFINITY PURIFIED FACTOR IX IN RABBITS: COMPARISON WITH COMMERCIAL CONCENTRATES
Authors: Smith K J
Published: June 2019

Commercial concentrates (CC) of vitamin K dependent coagulation factors may cause thrombosis or coagulation factor consumption while more highly purified (17 U/mg) factor IX (IX) concentrates do not seem to be thrombogenic (Menache et al, Blood 64:1220, 1984). Monoclonal antibody (MAb) immunoaffinity purified IX of high specific activity from CC or recombinant factor IX sources may also improve therapy. In this report, 400 mg of Affigel-10 linked A-7 MAb was used to bind factor IX in the presence of metal ions (20 mM MgCl2). Elution of IX was with 20 mM EDTA. Thrombogenicity of CC and IX prepared from CC by MAb immunoaffinity was tested. A CC which was thrombogenic in the stasis thrombosis assay (CC #1) produced large thrombi at doses of 50, 50, and 100 U/kg while none were seen with the IX produced from this CC at doses of 106 and 234 U/kg. A heparin treated CC (CC #2) which was not thrombogenic in the stasis-thrombosis assay at doses of 100 U/kg was infused in 4 rabbits at 100 U/kg and platelets, fibrinogen, AT-III antigen, and factors IX, V, and VIII were monitored for 5 hours post-infusion. Immunoaffinity IX from this CC was infused in 4 rabbits at 214-243 U/kg for comparison. Mean platelet count decrease was 20% in CC group and 8% for the IX group. Mean factor V and VIII decreased 26 and 36% respectively with CC while no decrease was seen in the IX rabbits (p < .05). Fibrinogen values and AT-III did not differ for IX or CC groups. Mean factor IX activity at 1 hour increased 1.6 fold for CC and 3 fold for IX. Yields for IX purification were 80 and 85%. Clotting activity was 143 and 101 U/mg and antigen was approximately 200 U/mg. Purification was over 90 fold by MAb immunoaf f inity. There was no detectable factor II, VII or X activity in MAb purified IX. Non-activated PTT was greater than 200 seconds for CC #1 and 158 seconds for CC #2. Column capacity was at least 150 mg. These results demonstrate that factor IX is not the thrombogenic component of some CC. Also, IX prepared by MAb immunoaffinity may have therapeutic advantage for patients at risk for thrombosis and adverse effects of contaminating proteins in commercial concentrates.
TYPE IIB VON WILLEBRAND DISEASE WITH CHRONIC THROMBOCYTOPENIA : BENEFICIAL EFFECT OF CRYOPRECIPITATE SUPERNATANT INFUSION ON PLATELET COUNT AND BLEEDING
Authors: Derlon A , Le Querrec A , Lebrun E , Tobelem G , Thomas M
Published: June 2019

As we previously described, plasma infusion increased platelet count (PC) in four patients with IIB von Willebrand disease with severe thrombocytopenia. In a sixty years old patient in the same family, with chronic thrombocytopenia (PC = 30 000/ml) associated to an absence of large von Willebrand Factor multi-mers (vWF) in plasma, we successfully treated :

1° A gastrointestinal bleeding episode with fresh frozen plasma infusion (15ml/Kg/day).

2° Three months later a severe epistaxis with cryoprecipi-tate supernatant (15ml/Kg/day).

During these bleeding episodes, the efficiency of these two treatments on the PC could be ascertained according to the following figure

We observed after ten days of these two treatments the following biological effects : a normalisation of vWF cross immunoelectrophoresis, of ristocetin induced normal platelet aggregation by patient's plasma, and of patient's plasma vWF binding to control platelets.

In conclusion a factor appears to be present in both fresh frozen plasma and cryoprecipitate supernatant which prevents the abnormal binding of von Willebrand Factor (in this IIB von Willebrand disease) to the patient's platelets.
A VARIANT OF VON WILLEBRAND'S DISEASE (vWD) WITH IIB-TYPE MULTIMER PATTERN IN THE ABSENCE OF ENHANCED PLATELET AGGLUTINATION
Authors: Cottrell S E , Wensley R T , Burn A M , Delamore I W
Published: June 2019

A 62 year old man with vWD has suffered from repeated episodes of melaena - his son and daughter have inherited the disorder with few symptoms so far. Laboratory findings in them include consistently prolonged bleeding times, normal factor VIII coagulant activity and decreased ristocetin cofactor activity. Levels of von Willebrand factor (vWf) antigen were lower measured by immunoradiometric assay than by Laurell immunoelectrophoresis. Analysis of vWf structure by SDS agarose gel electrophoresis showed loss of only the large multimers in plasma and the triplet structure of the smaller multimers showed sub-bands more intense than normal. However, the platelets contain the whole series of multimers with a similar pattern to normal. This was suggestive of type IIB vWD but agglutination of the patients' platelet-rich plasma with low concentrations of ristocetin was not enhanced. Agglutination was reduced compared to normal platelet-rich plasma at a final ristocetin concentration of 1 and 2 mg/ml and absent at a final concentration of 0.5 mg/ml. Platelet-type vWD was eliminated as addition of normal plasma or cryoprecipitate to patient's platelet-rich plasma did not produce spontaneous aggregation.

The patient has never had thrombocytopenia. We feel that this family demonstrates further the heterogeneity of vWD.
HEAT-TREATED FACTOR VIII CONCENTRATES IN VON WILLEBRAND'S DISEASE AND RELATED DISORDERS: STUDIES IN PLATELET-TYPE VON WILLEBRAND'S DISEASE
Authors: Takahashi Hoyu , Tatewaki Wataru , Nagayama Reizo , Hanano Masaharu , Takizawa Shin-ichiro , Shibata Akira
Published: June 2019

Cryoprecipitate has proved to correct the hemostatic defects in von Willebrand1s disease (vWd). However, recent studies have revealed that transmission of the AIDS retrovirus (HIV) occurs through exposure to blood products including cryoprecipitate. Treatment with heat-treated factor VIII concentrates may have certain advantages over treatment with non-heated products, if these preparations are efficacious in vWd and related disorders. We investigated the multimeric compositoin of von Willebrand factor (vWf), contents of vWf antigen (vWf:Ag) and ristocetin cofactor activity (RCof) in the heat-treated factor VIII concentrates and cryoprecipitate, and their capacity to directly induce aggregation of platelet-type (or pseudo-) vWd platelets in vitro. The vWf multimers were visualized by a newly developed, immuno-enzymatic staining of the gel, following a discontinuous SDS-agarose gel electrophoresis. The RCof/vWf:Ag ratio was around 1.0 in cryoprecipitate, and ranged from 0.19 to 0.96 in factor VIII concentrates. Among four commercially available concentrates studied, Haemate P contained the most high-molecular-weight multimers of vWf and the highest RCof relative to vWf:Ag, and induced the aggregation of platelet-type vWd platelets at the lowest concentration. When infused into a patient with platelet-type vWd, Haemate P (144 units vWf:Ag/kg body weight) shortened the prolonged bleeding time and caused spontaneous platelet aggregation in vitro with a mild diminution of platelet count (from the preinfusion value of 183,000μl to 139,000μl at 5 minutes). These results indicate that some of the heat-treated factor VIII concentrates contain the high-molecular-weight vWf multimers and may provide a safer, yet still effective, treatment for platelet-type vWd, and possibly for various types of vWd as well.
EFFECTS OF DDAVP INFUSION ON FVIII/vWF IN THREE CASES OF ACQUIRED TYPE II VON WILLEBRAND'S DISEASE
Authors: GOUDEMAND J , CARON C , JUDE B , PARQUET A , MAZURIER C
Published: June 2019

The response to a single intravenous infusion of DDAVP (0.4 μg/kg) was studied in 3 patients with acquired type II von Willebrand's disease associated with monoclonal gammopathy (2 cases) or chronic lymphocytic leukaemia (1 case). The Simplate bleeding time (BT) was prolonged to 10-26 min, FVIII:C, vWF:Ag (ELISA) and vWF:RiCof activities were respectively decreased to 0.15-0.48, 0.18-0.30 and 0.06-0.48 U/ml. Circulating inhibitor to FVIII/vWF were demonstrated in the 3 cases. Plasma vWF multimeric analysis was performed by SDS-agarose, electrophoresis and densitometric patterns were recorded. In that way, each well-separated multimeric unit can be expressed as a percentage of the total area under the curve representing the total vWF. Whereas the large (slower-moving) multimers (numbered 6 and more) represented 52.8-63.2 % of the total vWF in normal plasma (n = 9), they could account only for 12.5-28.7 % of the total vWF in the patients' plasma. In addition, a progressive decrease in the relative intensity of the multimers as their size increased, in a similar manner as is seen in type lie vWD, was demonstrated. The triplet structure seen in normal plasma was undetectable in 2 patients but present in the 3rd one.

Following infusion of DDAVP, the prolonged BT was normalized for 2 hr in 2 patients and transiently shortened in the 3rd one. FVIII:C, vWF:Ag and vWF:RiCof activities were increased to normal levels and remained higher than 0.5 U/ml for respectively 4-24 hr, 3-13 hr and 2-4.5 hr. The multimeric pattern could be considered as completely normalized in 2 patients and greatly improved in the 3rd one. The triplet structure was apparent in the 3 cases. The vWF multimeric structure returned to the preinfusion pattern by 5 hr in 2 patients and approximately 10 hr in the last one. These results demonstrate that endothelial cells from these patients are able to synthesize and release vWF with a full complement of multimers. Furthermore they show that the loss of the larger vWF multimers occurs after vWF has been released into the circulation.
FACTOR VIII:C (F VIII:C) AND VON WILLEBRAND FACTOR ANTIGEN (vWFag) IN RENAL TRANSPLANT RECIPIENTS IMMUNOSUPPRESSED WITH CYCLOSPORIN A (CyA)
Authors: Huser B , Lämmle B , Tran T H , Mihatsch M J , Thiel G , Duckert F
Published: June 2019

In 17 consecutive cadaveric kidney transplant recipients F VIII:C and vWFag were repeatedly determined before transplantation and during 4 months thereafter. Graft biopsy was performed in 12 patients for deterioration of renal function. F VIII:C was determined by a one stage clotting assay using F VIII:C deficient substrate plasma. vWFag was assayed by electroimmunoassay using specific rabbit anti-human vWFag antibodies. Results of F VIII:C and vWFag are expressed referring to NHP as 100%.

Results: 2/17 patients lost their graft due to irreversible vascular rejection, 2/17 patients had reversible vascular rejection, 2/17 patients developped glomerulonephritis, 6/17 patients showed acute or chronic CyA nephrotoxicity. In 5/17 patients graft biopsy was not necessary. Despite normalisation of renal function (serum creatinine levels<150umol/L) in 9 out of 17 patients F VIII:C (239 ± 66% to 408 ± 74%, mean ± SD) remained elevated in all 17 patients. vWFag (181 ± 29% to 454 ± 84%) was normalised in only 2 out of 17 patients. CyA dosis and CyA blood levels were not correlated with F VIII:C and vWFag. All 4 patients with histological vascular rejection, both patients with later developping glomerulonephritis and 3 out of 6 patients with later developping CyA nephrotoxicity showed F VIII:C/vWFag quotients > 1 (1,1 to 1,3) Four out of 5 patients with well functioning graft had F VIII:C/ vWFag quotients consistently <1 (0,68 to 0,92).

Conclusion: 1. The elevated F VIII:C and vWFag levels in chronic renal failure are not normalised during 4 months of observation despite normalisation of renal function by transplantation and immunosuppression with CyA.

2. A quotient F VIII :C/vWFag < 1 may indicate a good prognosis for kidney allograft function in the absence of CyA nephrotoxicity whereas later developping graft rejection or glomerulonephritis were associated with F VIII:C/vWFag>1.
CHANGES IN PLASMA t-PA, PAI AND FACTOR VIII FOLLOWING I. V. AND S. C. INJECTION OF DDAVP IN HEALTHY VOLUNTEERS
Authors: MacGregor I R , Roberts E , Prowse C V , Booth N A , Broomhead A , Litka P
Published: June 2019

DDAVP is used effectively to induce a haemostatic response in certain types of haemophilia A and von Willebrands disease, but the accompanying increase in fibrinolytic activity is an unwanted effect. Here we have quantified the extent and time course of changes in t-PA, its physiological inhibitor PAI and factor VIII moieties, using functional and immunometric assays, following i.v. and s.c. injection of DDAVP 0.4 ug kg-1 in 6 normal volunteers in a double blind crossover study.

T-PA, factor VIII and vWF values increased after dosing and the area under the response versus time curve was higher after i.v. than s.c. injection. Conversely levels of PAI activity decreased, with lower minimum values for i.v. than s.c. and a correspondingly lower area under the response curve for the former route. In several subjects PAI activity fell to zero during the sampling period. The times to the first occurrence of maximum response were determined. The median values for factor VIII were 40 and 90 min for i.v. and s.c. respectively. For vWF they were 60 and 80 min. For t-PA activity they were 20 and 60 min indicating that the release of factor VIII and vWF was delayed compared with t-PA activity. PAI-1 antigen was assayed by ELISA to investigate clearance of PA inhibitor and was compared with changes in PAI activity during the infusion of DDAVP and after a subsequent venous occlusion.

The results indicate that fibrinolytic and haemostatic responses follow different time courses from each other, both after i.v. and s.c. administration of DDAVP, that are reproducible from subject to subject. The use of specific assays has permitted determination of the fibrinolytic response to DDAVP to be assessed in terms of changes in pro and antifibrinolytic components.
VIRUS SAFETY OF ANTITHROMBIN III CONCENTRATE KYBERNIN P - APROSPECTIVE CLINICAL TRIAL
Authors: Clemens R , Wirl G , Velten C , Röthig H J
Published: June 2019

In 13 healthy male volunteers a prospective clinical trial was performed to evaluate virus safety of the antithrombin III concentrate Kybernin P in regard to hepatitis B, non-A/non-Band HIV transmission. As the volunteers were participants of a pharmacokinetic study they received either a fixed dosage of 1000 units Kybernin P asbolus injection or a dosage of 50 units per kg body weight as short-term infusion. Two different batches of Kybernin P were used.

Whereas in all 13 volunteers virussafety in hepatitis non-A/non-B and HAV transmission could be monitored, only those volunteers who were not vaccinated (n=3) against hepatitis B or who had no protecting antibodies of anti-HB type despite vaccination (n=3)were to be included in the hepatitis B monitoring.

All 13 volunteers were followed-upfor 1 year according to the standardsof the International Committee on Thrombosis and Hemostasis (ICTH). For detection of a potential hepatitis non-A/non-B transmission transaminases (AST, ALT) were determined in biweekly intervals during the first 6 months of the observation period and thereafter in monthly intervals. Hepatitis B seromar-kers as well as anti-HIV wereassessed bimonthly. Furthermore, all volunteers were clinically examined at every follow-up.

None of the 13 volunteers revealedan increase of transaminases to the 2.5 fold of the upper normal level which is considered to be the borderlinelevel for hepatitis non-A/non-B diagnosis. Furthermore, in none of the volunteers a seroconversion for hepatitis B or HIV could be detected. Thus, Kybernin P is to be considered as hepatitis B, hepatitis non-A/non-B and HIV safe.
PHARMACOLOGIC INEQUIVALENCE OF LOW MOLECULAR WEIGHT HEPARINS
Authors: Fareed J , Walenga J M , Hoppensteadt D , Emanuele R N , Racanell A
Published: June 2019

Compared to unfractionated heparin, low molecular weight heparins (LMWHs) have been found to exhibit marked variations in in vitro effects due to variations in molecular weight and structure. Moreover, when the in vitro potency of these agents is equally adjusted bypharmacopeial assay (current and proposed) wide variations in the in vivo responses have been noted. These variations were strongly dependent on the route of administration. Utilizing defined animal models, a systematic comparative study of the in vivo responses of seven commercial LMWHs was undertaken. Choay Fraxiparine (CY 216} Choay CY 222, NovoLHN, Kabi Fragmin, Opocrin 2123 (OP), Hepar RD 11885 (RD), Pharmuka Enoxaparin (PK) and Choay porcine mucosal heparin (PMH) were tested in identical settings at equigravimetric dosages. The graded results are given in the following.

Wide variations in the in vivo pharmacologic and toxicity responseswere noted suggesting that different LMWHs are not bioequivalent at equigravimetric levels. When these responses were expressed in anti-factor Xa or pharmacopeial potency, these differences were further magnified. The clinically reported dosimetric and safety problems may be minimized by profiling LMWHs in defined in vivo test systems to optimize their safety/efficacy ratio.
AMBULANT MONITORING OF HEPARIN AND HAEMOSTASIS DURING VASCULAR SURGERY
Authors: Porte R J , de Jong E , Knot EA R , de Maat MP M , Terpstra O T , van Urk H , Groenald TH N
Published: June 2019

Heparin has been used to prevent thranbotic events in vascular surgery for more than 45 years. Heparin-activity monitoring has been advocated, but is not usually performed routinely. A direct method for measuring heparin anti-Xa activity with a chromogenic substrate is difficult to perform during surgery for logistic reasons or lack of suitable equipment. The use of this assay, however, could give us a better insight in the kinetics of heparin during vascular surgery. To use this heparin-activity assay during vascular surgery in ccmbinatian with clotting assays and a antithrcmbin III-assay, one should be able to perform both kinetic and end-point methods in a rapid, simple and reproducable manner. Therefore, in this study we tested the FP-910 coagulation analyser (Labsysterns), in which both methods can be assayed. It was used during 20 consecutive abdominal aorta reconstruction operations, in which a standard dose of 4-5,000 IU heparin was given intravenously. Bloodsairples were taken at several intervals, APTT, PT, ThT, fibrinogen, AT-III and heparin could be assayed within 30 min. after a bloodsanple was taken, with good intravariations (1.8%, 3.3%, 3.9%, 7.5%, 4.9% and 5.0%, ersp.) and intervariations (5.0%, 4.5%, 3.8%, 11%, 8.9% and 6.1%, resp.). Heparin activity alone could be measured within 10 min. The preoperative sanples shewed no abnormalities. Heparin activity response 5 min. after injection showed a wide variation (0.2-2.8 IU/ml) and this was also seen in the individual heparin elimination rates. In one case a thranbotic complication occured during a period of low heparin activity (0.1 IU/ml). In another patient a combined decrease of fibrinogen and AT-III, to 30% and 37% resp, of the initial value was seen. These results showed that, during vascular surgery, a close monitoring of heparin activity with a anti-Xa activity assay, together with other haemostasis parameters is necessary, and possible in a rapid and simple way, using the FP-910 coagulation analyser.
STUDIES ON THE MECHANISM OF THE ANTITHROMBOTIC ACTION OF A CHEMICALLY SYNTHESIZED HEPARIN PENTASACCHARIDE
Authors: Bara L , Walen-ga J M , Petitou M , Samama M , Fareed J , Choay J
Published: June 2019

A chemically synthesized heparin pentasaccharide (PS) (Instir tut Choay, Paris, France) has been reported to exhibit an antithrombotic action in a rabbit stasis induced thrombosis model, in an IV dose range of 25-200 ug/kg (0.5 to 5 ug/ml circulating concentrations) . Ex vivo plasma analysis from treated animals revealed expected angi-factor Xa activity byboth amidolytic and coagulant (Heptest®) methods. Nodirect inhibitory effect against factor IIa by amidolytic or coagulant methods was observed. Global anticoagulant activities were not found by PT and APTT methods; however, a hypocoagulable thrombelastographic pattern was demonstrated for native whole blood. Platelet activation remained unaffected at the antithrombotic dosages of PS. An attempt was made to more specifically elucidate the anti-factor Xa mediated antithrombotic mechanism of action of PS. The effect of this agent was studied in several thrombin generation assays in human and rabbit plasmas supplemented in vitro with a 0-5 ug/ml concentrations of PS. The following systems were used: 1) activated prothrombin complex (FEIBA®) , thromboplastin-Ca+2/synthetic substrate; 2) prothrombin complex concentrate (Konyne), thrombo- plastin-Ca+2/synthetic substrate; 3) cephalin-ellagic acid-Ca+2/ synthetic substrate (modified Fischer method); 4)cephalin-Ca / synthetic substrate (modified Ofosu method); 5) FEIBA /FPA re- dioimmunoassay; 6)whole blood prothrombin consumption/clot; and 7) cephalin-Ca+2/clot (Hicks-Pitney method). All assays produced a concentration-dependent effect to a 35-50% inhibition of generated thrombin with 4-5 ug/ml PS concentrations. Assay #4 also revealed that although PS inhibited the generation of thrombin, the thrombin that was generated was inhibitedby natural plasma proteins at normal kinetic rates. In ex vivo studies similar concentration-dependent inhibition of thrombin generation was observed using assays #3 and #4. These results indicate that the inhibition of induced venous stasis thrombosis is associated with the inhibition of thrombin generation byPS without a direct inhibition of pre-formed thrombin. Furthermore, these results indicate that thrombotic events may be controlled at pre-thrombin coagulation stages by agents with sole anti-factor Xa activity.
A TRIAL OF SUBCUTANEOUS VERSUS INTRAVENOUS ADMINISTRATION OF LOW MOLECULAR WEIGHT (LMW) HEPARIN AND UNFRACTIONATED (UF) HEPARIN IN THE TREATMENT OF ESTABLISHED DEEP VENOUS THROMBOSIS (DVT)
Authors: Parker C J , Huber D E , Hedges A R , Kakkar V V
Published: June 2019

In a randomized clinical trial of 100 patients, the in vivo antithrombotic effects of a subcutaneously administered LMW heparin fraction (CY216) used in the treatment of established DVT, was compared with UF heparin administered by either intravenous or subcutaneous routes.

Venograms were used to make the initial diagnosis, and efficacy of treatment was assessed by a repeat venogram done on day 6. Comparison of the venograms were done blind by an expert radiologist.

Patients were randomized to one of three groups: Group 1 received subcutaneous CY216; Group 2 received subcutaneous UF heparin: Group 3 received continuous intravenous UF heparin. Random patients from each group had detailed haematological tests consisting of twicedaily KCCT and anti-Xa levels. Extension of thrombus occurred in significantly morepatients receiving intravenous heparin than subcutaneous heparin (p-0.02).

There was no difference between the two subcutaneousgroups. There were no haematological complications.

We conclude that subcutaneous administratiyon of heparin is the treatment of choice in the treatment of DVT.
ARE THE SYMPTOMS OF THE POST PHLEBITIC LIMB ALWAYS POST THROMBOTIC
Authors: Parker C J , Huber D E , Hedges A R , Kakkar V V
Published: June 2019

The term ‘post phlebitic syndrome’ implies a previous history of deep venous thrombosis. To test the validity of this assumption, 106 patients who had routine post-operative bilateral ascending venography following total hip replacement were reviewed five years later.

Patients were assessed clinically for symptoms and signs of the ‘post phlebitic syndrome’ (pain, swelling, induration, pigmentation, ulceration and varicose veins). Haemodynamic changes were assessed by foot volumetry and an objective score was derived by computer analysis

Thirty patients (28%) had post-operative DVT. 50% of all patients had at least one symptom or sign of the post phlebitic limb. Pain was present in 9 limbs; swelling in 13; induration in 18; pigmentation in 55, and varicose veins in 53 limbs.

At five years there was no significant difference in the incidence of symptoms, signs or haemadynamic changes of the post phlebitic syndrome between limbs with or without a previous DVT. We conclude that deep vein thrombosis is not the only factor involved in the aetiology of the post phlebitic limb.
ALTERATIONS IN THE COAGULATION AND FIBRINOLYTIC SYSTEMS AS PREDISPOSING FACTORS IN THE DEVELOPMENT OF DEEP VENOUS THROMBOSIS (DVT)
Authors: Tengborn L , Wallmark A
Published: June 2019

From Jan. 1982 to Jan. 1987, 1213 patients were investigated either because of thrombotic episode(s) or thrombosis heredity. Diagnosis of DVT was confirmed by phlebography in 567 cases. Patients were first examined at least three months after an acute episode.

Methods. Anti thrombin (AT) and plasminogen were assayed, using chromogenic substrates S-2238 and S-2251 , respectively, followed if values were low by immunochemical assessment. Furthermore, fibrinogen, thrombin and reptilase times, APTT, P…P were assessed. Only in the latter study period was protein C activity determined (129 cases). Fibrinolysis was assayed on fibrin plates after 20 min venous occlusion of the arms. In 1984 only, plasminogen activator inhibitor of endothelial cell type (PAI 1) was measured in most DVT patients (n=75).

Results. DVT first occurred by the age of 45 in 337 patients, of whom five had AT deficiency (four classic and one abnormal), one had low and another abnormal plasminogen), one had abnormal fibrinogen, seven had lupus anticoagulants (LA), and 72 (21%) had decreased lysis on fibrin plates. Defective fibrinolysis was re-investigated in 42 patients; at check-up 13 were found to have normalised. Of the 75 patients from 1984, fibrinolysis on fibrin plates was normal in 50 cases, of which PAI 1 was normal in 44 and increased in six; of the remaining 25 patients from 1984, fibrin plate activity was decreased, PAI 1 was normal in five cases and increased in 20.

DVT first occurred after the age of 45 in 230 patients, of whom none had pathological AT or protein C, five had LA, and one had abnormal plasminogen; of the 51 (22%) patients found to have defective fibrinolysis, 29 were re-investigated at check-up and 14 of them found to have normalised.

Conclusion. Alterations in coagulation inhibitors are rare in patients with DVT. A more frequent finding, although intraindividual fluctuations occur, is defective vessel wall fibrinolysis.
LONG TERM EFFECTS OF TICLOPIDINE ON FIBRINOGEN AND HAEMORHEOLOGY IN PATIENTS WITH PERIPHERAL ARTERIAL DISEASES
Authors: Palareti G , Torricelli P , Poggi M , Fortunato G , Oca G , Coccheri G
Published: June 2019

Fibrinogen (Fgn) and haemorheologic parameters were serially measured in 44 pts (38 males, age 59+/−7) with claudicatio intermittens due to peripheral arterial obliterative disease (PAOD) treated for 24 months with Ticlopidine (T group, 250 mg b.i.d. p.os), or placebo (P group), in a double blind randomized controlled study, part of a larger clinical trial. Fgn (immunodiffusion), haematocrit (Ht, micromethod), whole blood viscosity (BV, Contraves LS 30, at 37 C) at high (94.5) and low (0.2 sec-1) shear rates (s.r.) and plasma viscosity (PV, Contraves LS 30) were measured three-monthly. The data were evaluated by means of ANOVA and Student's t test. The values in groups T and P did not differ at baseline except for greater BV at high s.r. in group P (p< 0.05). During the observation period circannual variations appeared especially in group T, where summer-time values for most parameters (Fgn, Ht, PV, low s.r. BV) were lower (significance from p<0.05 to <0.01) than the correspondent winter-time values, while a similar pattern was observed in group P only for PV (p<0.01). Fgn (p<0.05), Ht (p<0.01), and low s.r. BV (p<0.001) were significantly lowered in group T versus group P but only in the summer months (after about 1 year of treatment). In conclusion, this study proves that long-term Ticlopidine treatment in PAOD is associated with significant lowering of fibrinogen and “improvement” in haemoreologic tests although limited to the summer observations. Ticlopidine while positively interfering with haemorheology in PAOD may not counteract other mechanisms of rheologic deterioration probably occurring during winter. Long-term evaluation of drug effects should therefore take into account spontaneous or drug-enhanced seasonal changes of the investigated values.
NOVEL TECHIQUES FOR QUANTIFICATION OF RBC-SHAPE (RS) AND SHEAR INDUCED RBC ELONGATION (SIRE): APPLICATION FOR ANALYSIS OF DRUG INDUCED ALTERATIONS
Authors: Artmann G , Grebe R , Wolff H , Degenhardt R , Schmid-SchÖnbein H
Published: June 2019

In the past, red cell resting shape could only be assessed by subjective scaling, red cell deformability by a variety of rheological tests that are extremelydifficult to standardize and which all subject the RBC to high deforming forces. None of the latter have been accepted as reference in haematology, haemorheologyor pharmacology. A recent development from our group now allows objective, numerical analysis of red cell membrane curvature (i.e. the echinocytic or stomatocytic deviation from the discocytic resting shape) by a tangent count procedure in optical sections through freely suspended, randomly oriented RBC: (Grebe et al. Biorheology 22(6), 1985). Also, the deformation of point attached erythrocytes under the influence of extremely low shear stresses (0.05 Pa to 0.5 Pa, ARTOANN:Clin. Hemorheology 6, 1986), which are at least two orders of magnitude lower thanthat in any routinely available filtration method allows for the first time to model in vitro the extreme low flow states that occur in severe forms of haemodynamic insufficiency. These two methods in combination are ideally suited for routine tests of drug effects on normal human RBC: the drug action on RS can be monitored continuously during the action of drugs in the suspending medium; likewise, RISA can be recorded automatically on one population of adherent RBC while altering the composition and the drug concentration in the superfusate. The two methods were applied in combination to test rheological and membranological effects of two distinctly different compounds, namely Bencyclan (Bencylan-Hydrogen-Fumarate) and Vinpocitin (Aethyl vincamin) in normal cells and in cells after exposure to "stress conditions", i.e. hyperosmolarity and lactacidosis. Both olrugs given to n o r m a 1 RBC produce stomatocytosis in a done dependent fashion (1-100 uMolar). At shear stresses above o.6 Pa, the RISA is identical to controls, but is oxmsiderably less pronounced at lower shear stresses (T < 0.2 Pa). Thus, drugs of completely olifferent pharmacological action produce clear cut rheological effects on RBC in the micrcmolar concentration range; the combination of methods employed opens new possibilities for the systematic development of haemorheologically active drugs.

Supported by DFG:Grant Gr 902/1-1
SYSTEMIC FIBRINOLYSIS AND PLASMA VISCOSITY REDUCTION: A SECOND BENEFIT
Authors: Moriarty A J , Hughes R , Nelson S D , Balnave K
Published: June 2019

The aim of this study on a small cohort of patients (N = 30) with acute myocardial infarction (AMI) receiving systemic streptokinase (STK) thrombolytic therapy was to measure the decrease in plasma viscosity concomitant with fibrinogen depletion.

Serial measurements of plasma viscosity, plasma fibrinogen and haematocrit were undertaken in treatment and control groups at times t = 0, 1 hour, 6 hours, 12 hours, 24 hours and daily thereafter over a period of six days. Viscosity was measured at 25°C using a Wells-Brookfield cone/plate digital viscometer, and fibrinogen determination was by the method of Clauss.

Results indicated a mean percentage drop in plasma viscosity of −17% in the STK-treated group with a maximum drop in any one patient in excess of −30%. This contrasted with a mean viscosity rise of almost +20% in the control group with a maximum rise in any one patient of almost +44%, parallelling the rise in plasma fibrinogen as an acute phase reactant.

Correlation studies between viscosity and plasma fibrinogen were strongly positive with mean r-values of 0.74 and 0.66 respectively. Interestingly, mean plasma viscosity in the STK-treated group was still reduced by −5% at 6 days post-therapy.

The conclusions drawn from these data are that the benefit of systemic STK treatment in AMI may in part be due to reduced myocardial workload and oxygen consumption at a critical time through afterload reduction, and improved collateral circulation and microcirculation consequent on the reduced plasma viscosity. Clearly there may be implications for newer tissue-specific agents which do not have a substantial systemic effect.
THE PREVELANCE OF ANTIPHOSPHOLIPID ANTIBODIES, BY ELISA TECHNIQUE, IN PATIENTS WITH THE LUPUS ANTICOAGULANT
Authors: Brien W , Denome G , O’Keefe B
Published: June 2019

Patients with the Lupus Anticoagulant and/or anticardiolipin antibodies have been reported to be at increased risk of thrombosis and miscarriages. It has been proposed that the lupus anticoagulant is an antiphospholipid antibody.

We evaluated 16 patients with the lupus anticoagulant for the presence of antiphospholipid antibodies. The lupus anticoagulant was documented by the presence of an abnormal APTT, abnormal mixing studies, positive tissue thromboplastin inhibition test and positive platelet neutralization test.

Plasma from each patient was assessed for the presence of anticardiolipin, antiphosphatidylserine and antiphosphatidyl-glycerol antibodies by ELISAtechniques. As a control, a neutral phospholipid phosphatidylethanolamine was used. A positive result was established when a delta value of lipid minus control was greater than 3SD compared to a normal population (20 pt.).

Using three different patient dilutions, positive results were obtained in 10/16 pt. for anticardiolipin, 11/16 pt. for antiphosphatidylserine and 5/16 pt. for antiphosphatidyl-glycerol antibodies. Three patients were negative for all lipids. If a neutral phospholipid was not used and a delta volume not obtained, 15/16 patients would have had positive results.

Our results suggest 1) Not all patients with the Lupus Anticoagulant have antiphosphilipid antibodies by ELISA technique. In evaluating patients with thrombosis and/or miscarriages, both tests should be performed.

2) Anticardiolipin antibodies are not present in all patients and with a panel of other negatively charged phospholipids more positive results are obtained. 3) A neutral lipid should be used as a control for non-specific binding of antibody and delta values obtained to see if the results obtained is truly against the negatively charged lipid.
PLASMA FREE PROTEIN S AND OESTROGEN ADMINISTRATION
Authors: Melissari E , Scully M F , Parker C , Hedges R , Kakkar V V
Published: June 2019

It has been shown that protein S has a cofactor role for the anticoagulant and fibrinolytic activity of activated protein C (APC) and that the free protein S fraction is the main cofactor for the APC. Here we report free protein S levels from three groups of women on oral oestrogen. Group A: Five post menopausal women (36-48 years), on oestradiol valerate (as hormone replacement therapy after hysterectomy-oophorectomy) mean free protein S was 62.8% ( 2SD below normal adult mean). Group B: Seven young women (20-25 years) on the contraceptive pill. Two had normal levels of free protein S (100% and 125%), whereas the other five were 30%, 29%, 50%, 60% and 64% of normal. Group C: Two young women. The first, a 25 year old, insulin-dependent diabetic woman developed severe thromboembolic disease shortly after initiation of oral contraception. The second, a 21 year old woman with congenital, moderately decreased plasminogen (PLG) (activity and antigen 45% of normal), had a severe ileofemoral deep venous thrombosis about 8 months after initiation of oral contraceptive (her free protein S levels were twice found to be 60% of normal). In both cases, family members with reduced levels of PK and PLG respectively were free from any thromboembolic disease and had normal protein S levels. Since an association between oral contraceptive use and incidence of venous thromboembolism without predisposition has been consistently observed in case-control and cohort Studies, we conclude that oestrogen may reduce the plasma free protein S concentration and increase thrombophilia.
THE CLINICAL SPECTRUM OF PROTEIN C DEFICIENCY IN A LARGE NEW ENGLAND KINDRED
Authors: Bovill E , Bauer K A , Callas P , West B , Dickerman J D
Published: June 2019

A family with a high incidence of venous thromboembolism over six generations has been investigated. Medical histories have been obtained on 136 of the 368 members of the kindred which allowed assignment of individuals into positive, equivocal, or negative categories with respect to their thrombotic manifestations. Protein C levels were determined by antigenic assay. Patients with protein C levels less than 66% of normal (mean, minus 2 SD) were classified as having protein C deficiency.

The mean age of onset of thrombotic manifestations in the 10 protein C deficient patients with a positive history was 33.8 ± 16.4 years while the mean age of the deficient group with a negative history was 22.0 ± 10.9 years. We conclude that there exists a striking association between thromboembolic disease and protein C deficiency within this large New England kindred.
Clinical Relevance of Blood Glucose Concentration and Hyperglycemia Management in Neurocritical Care Patients
Authors: Bilotta Federico , Qeva Ega , Prete Anna , Pugliese Francesco
Published: June 2019

In patients admitted to a neurocritical care (NCC) unit, management of blood glucose concentration (BGC) is a challenging clinical task. Several studies have shown that episodes of hypo- and hyperglycemia and high BGC variability are associated with poor short- and long-term outcomes. Optimal BGC target-range and BGC management in NCC patients have dramatically evolved in the past decades and new insulin infusion and adequate nutrition protocols are now available. The aim of this narrative review is to report the state-of-the-art on clinical relevance on BGC and hyperglycemia management in NCC patients.
Membrane Transports of Platelets Present in Plasma During Clotting and Lysis
Authors: Zuzel M. , Irving W. , Born G. V. R.
Published: June 2019

During blood clotting and subsequent fibrinolysis a major proportion of platelets survive as single entities. Platelets exposed to thrombin in vivo or in vitro survive normally in the circulation (Reimers et al., 1973, Brit. J. Haemat., 25, 675), in spite of having undergone a release reaction.

Our experiments were to find out whether clotting and lysis of plasma impair transport functions of platelet membranes. Human citrated platelet -rich plasma was incubated at 37° with thrombin plus fibrinolytic activator. Rapid clotting was followed by complete fibrinolysis within 3 min. By then about 60% of radioactive 5-hydroxytryptamine previously taken up by the platelets was released; this was accompanied by the loss of about 40% of platelet potassium which was subsequently re-accumulated. The initial velocities of uptakes of 5-hydroxytryptamine and adenosine were almost identical in platelets before and after clotting and lysis. Therefore, at least three energy-requiring transport processes in the platelet membrane were unaffected by the chemical events associated with clotting and lysis of platelet-rich plasma.
A High Titre Circulating Inhibitor of Human Factor V: Clinical, Biochemical and Immunological Features and its Treatment by Plasmapheresis
Authors: Grace G. S. , Wolf P.
Published: June 2019

A 44 year old male with a two month history of epistaxis and haematuria was admitted because of melaena. He had not received antibiotics or blood transfusions. Haemostatic studies showed a complete absence of Factor V activity and this was not corrected by the addition of an equal part of normal plasma. Incubation studies showed that the inhibitor was specific for human Factor V. Factor VIII and IX activities were low by one stage assay methods but were normal after the addition of bovine Factor V.

The titre of the antibody was very high; a 900 fold dilution causing a 50% reduction in the Factor V level of an equal volume of normal plasma on incubation at 37° C for 30 mins. The inhibitor appeared in the second protein peak on Sephadex G200 fractionation and was neutralized by antihuman IgG antiserum. Kinetic studies showed the inhibition to be first order with respect to Factor V and inhibitor.

The patient was treated with plasmapheresis on a IBM Celltrifuge. Four litres of plasma were removed on each of four occasions over a period of 14 days. The antibody titre dropped from 900 to about 100. Despite the addition of immunosuppresive therapy the antibody has persisted at a titre of about 200 and has now been present over 5 months. No anamnestic response occurred following multiple transfusions.
Immunochemical Studies on the NH2-Terminal “Disulfide Knot” and Related Structures
Authors: Kudryk B. , Blomback B.
Published: June 2019

A radioimmunoassay for the NH2-terminal portion of human fibrinogen (N-DSK) was recently used to measure cross-reactivity between several related fragments (Eur. J. Biochem. 46: 141–147, 1974). The results obtained showed that “cold” N-DSK and the NH2-terminal portion of plasmic Fragment E (“E-knot”) were strong competitors while Fragment E itself was a poor competitor in the system 125I-labeled N-DSK/anti-N-DSK. In this paper we report results from further experiments designed to study the basis for the observed immunological difference between Fragment E and “E-knot”. Using the N-DSK radioimmunoassay system we have now shown that formic acid denaturation alone cannot account for this difference. Additionally, intact mixtures of CNBr-treated Fragment E competed to the same degree as “E-knot”. The latter finding would suggest that there appears to be no interaction of the split COOH-terminal peptides (from Fragment E) with “E-knot” resulting in the “masking” of epitopes. However, at this point we cannot be certain that such “masking” of epitopes by these peptides does in fact occur in intact Fragment E.

In a related study we have also tested the cross-reactivity of a disulfide core fragment obtained from N-DSK by trypsin digestion. This fragment (DS-4) has an approximate molecular weight of 8,000 and has 4 disulfide bridges. The latter does not react on immunodiffusion using antisera to N-DSK. However, in the N-DSK radioimmunoassay system DS-4 showed very strong competition. Approximately 73% inhibition was observed at the 10 nmol level. The significance of these results with respect to the location of highly antigenic regions of N-DSK will be discussed.

(Supported by N.I.H. grant HL 09011–11.)
Characterisation of a Soluble High Molecular Weight e Fragment Released by Plasmin from Cross-linked Fibrin
Authors: Lane D. A. , Brasher M. , Kakkar V. V. , Gaffney P. J.
Published: June 2019

The heterogeneity of fragments released by plasmin degradation of cross-linked human fibrin has been examined. Six components (labelled 1–6) have been detected by PA gel electrophoresis and the two components (3 and 4) that make up the D dimer complex have been examined in relation to fragments D and E. Using two dimension Immunoelectrophoresis (with PA gel in the first dimension) and immunodiffusion, together with SDS PA gel electrophoresis of the fragments and their subunits, it has been shown that the more anodic component (band 4) of the complex is D dimer. Band 3, which is the recently reported high MW component (Gaffney et al., Clin. Sci. Mol. Med., in press) that reacts with antisera to fragments D and E, is shown to be a strong non-covalent association of D dimer and E. This observed heterogeneity of the components of fibrin digestion is significantly more complex than has been previously reported.
Factors Influencing the Binding of Radiolabelled Collagen to Blood Platelets
Authors: Maclntyre D. E. , Gordon J. L.
Published: June 2019

The binding of radiolabelle collagen to blood platelets has been investigated using a modification of the Ficoll density gradient centrifugation technique (Gordon and Dingle, 1974). The binding of 125I labelled neutral salt soluble rat skin collagen to rat platelets is maximal within 10 sec, increases with temperature (4°-37°), and with increasing pH (7.0–8.0). The extent of binding is similar in platelet-rich plasma anticoagulated with citrate (0.31% w/v final concentration in blood) heparin (5 IU/ml) or EDTA (0.1% w/v), and in washed platelet suspensions. The ability of collagen to bind to platelets is abolished by prior heating to 56° for 30 min, or by collagenase digestion (20 μg/ml, 30 min at 36° C). A range of known inhibitors of collagen-induced platelet aggregation were tested as potential inhibitors of binding. Aspirin (1.1 mM), PGEl5 (3 μM), glucosamine (1 mM) and Deoxyglucose (15 mM) in combination with Antimycin A (5 μg/ml) did not reduce the binding of collagen to platelets. Glutaraldehyde fixation of platelets (0.01%, 5 min at 20°) markedly reduced the extent of collagen binding, but binding to formaldehyde fixed platelets (5%, 30 min at 20°) was similar to controls.

This study indicates that collagen tertiary and quarternary structure are necessary not only for platelet aggregating activity, but also for the initial binding of collagen to platelets. Binding is independent of platelet metabolic activity and is not readily susceptible to pharmacological inhibition.

Gordon, J. L. and Dingle, J. T. (1974), J. Cell sci. 16; 157.
New Sinthetic Derivatives of Natural Cyclic Nucleotides: Their Effect on Platelet Behaviour
Authors: Pareti F. I. , Mannucci L. , Carrera D. , Mannucci P. M.
Published: June 2019

New sinthetic derivatives of natural cyclic nucleotides have been selected on the basis of their favourable characteristics after a preliminary screening of metabolic and cardiovascular effects in rats. The average hyperglycemic and cardiovascular effects of 8-substituted compounds were lower than those of 6- and 2’0-substituted derivatives despite a high resistance to phosphodiesterase. Four of the tested 8-substituted compounds inhibited platelet aggregation induced in PRP by a standard dose of ADP (3 μM). The most effective was 8-Bromo 3′-5′ GMP (50% inhibition of primary aggregation at 0.2–1 mM concentration; N6, 2’0 Dibutyryl 3–5′ AMP (DB-cAMP): 3–5 mM), which also inhibited 14C serotonin release at lower doses than DB-cAMP. ATP breakdown and hypoxanthine formation induced by exponsure of platelets to ADP was decreased in presence of the compound. Other effective 8-substituted derivatives were 8-mercapto 3–5′ GMP (0.8–1.5 mM), 8-methylmercapto 3–5′ GMP (5–8 mM) and 8-Bromo 3–5′ IMP (5 mM), whereas 8-Bromo 3–5′ AMP and 8-Piperidino 3–5′ AMP were ineffective.
Antibiotics, Platelet Aggregation and Electrophoretic Mobility
Authors: Houbouyan L. L. , Stoltz J. F. , Goguel A. F.
Published: June 2019

The in vitro influence of twelve antibiotics (representing the principal classes: β-lactam group, aminoglycosides, polypeptidic antibiotics, antimitotics, . . .) on the ADP-induced Platelet Aggregation, the Screen Filtration Pressure test and the liquid phase Electrophoretic Mobility (E. M.) has been studied. According to the results formerly obtained on the interference Penicillin G-platelets, the authors have mainly investigated the β-lactam group (Penicillin G, Ampicillin, Carbenicillin, Cefalotin, Metampicillin, Methicillin, Oxacillin, . . .). This group seems particularly interesting as an antiaggregating effect has been found out for most of them; a parallel decrease of the E. M. has been generally observed.

In attempts to elucidate the mechanism of physiochemical interaction with the membrane, the competitive effect of phospholipases and Penicillin G has been studied. The results seem to point out an interference of this antibiotic with the membrane phospholipids; this could be in accordance with the rheological data of Padefield and Kellaway. The interference with some other membrane groups has also been considered (carboxyl, phosphates . . .).

An in vivo study has also been realized on patients massively perfused with some of these antibiotics (especially Penicillin G, Carbenicillin . . .).

(This work was supported by Grant of the DRME (Biological Section).)