bluuebunny/crossref_metadata_2025 huggingface dataset:
Dibucaine-Activated Platelet Protease(S) Digests GPIb-EDTA Only Partially Inhibits
Authors: Coller Barry S
Published: July 2018

Local anesthetics have been reported to have several effects on platelets including: 1) inhibition of shape change, the release reaction and aggregation induced by several agents 2) dissolution of cytoskeletal structures and 3) activation of a protease activity which digests proteins of Mr 250,000 and 230,000. It was speculated that the last effect may be due to the Ca++-dependent protease previously described in platelets. I have recently observed that incubation of platelets with 1 mM dibucaine for brief periods of time (15-30 min) resulted in 50-100% enhancement of the initial slope of agglutination induced by either ristocetin or bovine von Willebrand factor (vWf) despite inhibiting the release reaction. If the platelets were formaldehyde-fixed after brief incubation, they also showed the enhancement (control 23 ± 3 U/min (mean ± SEM) and dibucaine 47 ± 6, p < 0.01, n = 7 for ristocetin and 36 ± 7 vs. 65 ± 5, p < 0.01, n= 6 for bovine vWf). Using these platelets, the ristocetin cofactor assay was more sensitive and yet equally specific. If, however, fresh platelets were incubated with dibucaine for > 1 hour, both ristocetin and bovine vWf-induced agglutination became progressively inhibited with almost total inhibition after several hours. This decrease in agglutination was associated with a progressive loss of a glycoprotein with the characteristics of GpIb (Mr 165,000 unreduced and 135,000 reduced) as judged by PAS-stained polyacrylamide gels. The presence of the platelet-impenetrable calcium chelator EDTA (10 mM) only partially prevented the decrease in agglutinability and loss of Gplb. I conclude that: 1. Short term incubation with dibucaine may improve the ristocetin cofactor assay. 2. A dibucaine-activated protease(s) appears to digest Gplb, probably accounting for the decreased agglutination with long incubations. 3. If operating from the outside of the platelet after leakage, at least some of the protease activity is not calcium dependent.
Effect Of Storage On Surface Glycoproteins And Cyto-Skeletons Of Platelets
Authors: Rao A K , Tuszynski G P , Knight L , Willis J , Beckett C
Published: July 2018

Platelets stored as concentrates (PC) at 22° C for 72 hours develop a functional defect in vitro tests. Alterations in membrane glycoproteins of platelets have been shown to effect platelet function. We have investigated the effect of storage on membrane glycoproteins (GP) and cytoskeletons (cyto.) of platelets. Gel filtered platelets from fresh PC were labeled with 125Iodine by Iodogen technique and gel filtered again to remove free iodide. Platelets were concentrated by albumin density gradient centrifugation, resuspended in autologous plasma and stored for 72 hours at 22° C. Aliquots of fresh and stored PC were solubilized with 2% sodium dodecyl sulphate (SDS) containing 5% mercaptoethanol and subjected to polyacrylamide gel electrophoresis (PAGE). In one experiment, separate aliquots of fresh and stored platelets were labeled and similarly analyzed. Gels were stained with Coomassie blue and subjected to autoradiography. Coomassie blue staining did not reveal major differences between fresh and stored platelets. Autoradiography revealed a decrease in the 170,000 dalton surface protein (GP-I) of platelets after storage. Triton insoluble cyto. of thrombin activated fresh and stored platelets were solubilized with SDS and analyzed by PAGE and autoradiography. Cytoskeletons from fresh PC revealed the presence of a 110,000 dalton surface protein (GP-III). However, cyto. from similarly treated stored platelets showed a markedly decreased amount of this protein. Thus stored platelets have decreased amounts of the 170,000 dalton surface protein (GP-I) along with decreased amounts of the 110,000 dalton protein (GP-III) associated with the cyto. of thrombin activated platelets. These changes may contribute to the functional defect reported in stored platelets.
Warfarin Induced Fibrinolysis: The Effect Of Recovery With And Without Vitamin K
Authors: Marsh N A
Published: July 2018

We have previously shown that fibrinolysis in the rat is enhanced by levels of warfarin administration sufficient to produce moderate anticoagulation. This effect is mediated largely by an increase in plasma plasminogen activator. Plasminogen levels are decreased and fibrin(ogen) degradation products raised confirming the presence of a systemic hyperfibrinolytic state. In order to investigate this phenomenon further we have measured fibrinolytic components in rats recovering from warfarin administration. Groups of male Hooded rats received 14 yg warfarin/100 g body weight /day by mouth for one to two weeks. The animals were then allowed to recover without further treatment or following a single 50 μg dose of vitamin K1. Euglobulin lysis time, one stage prothrombin time, plasma plasminogen activator (fibrin plate method), plasma plasminogen (caseinolytic method), plasma fibrinogen (clot weight method) and plasma fibrinolytic inhibitors were measured at intervals after the end of the warfarin treatment. In animals recovering without vitamin K prothrombin time returned to normal within one week. However fibrinolysis remained elevated with plasma plasminogen activator concentrations of more than twice the control value. Plasma inhibitor levels were depressed and fibrinogen levels elevated. After two weeks all fibrinolytic components had returned to normal. Following vitamin Kj administration a different pattern emerged; prothrombin time returned to normal within 24 hours but fibrinolysis was diminished. The latter effect was due to a marked increase in plasma fibrinogen and moderate fall in plasma plasminogen activator both of which contributed to a prolonged euglobulin lysis time. These results indicate that fibrinolysis and coagulation in the rat are not linked in a 'dynamic equilibrium' like that proposed for man. The enhanced fibrinolysis rather than being a result of the fall in vitamin K dependant clotting factors may be due to the direct action of warfarin. The 'fibrinolytic shutdown' following vitamin K remains unexplained .
Higher Efficiency In Clot Lysis Rate Of Circulating Plasminogen Activators After DDAVP Infusion Compared To That Of Activators Bound To Clots
Authors: Tran-Thang C , Bachmann F
Published: July 2018

Clot lysis rate, in presence or absence of vascular activator (VA) during clotting and in the surrounding medium, was studied. The mean fibrinolytic activity of plasma euglobulin fractions (EF) was 0,4 U UK/ml (measured on fibrin plates) in resting volunteers; it increased 6-8 fold ten min after the i.v. infusion of DDAVP (0,4 μg/kg). No activity was detectable in serum EF before and after DDAVP infusion, but, if plasma (10 mM citrate - 5 mM EDTA) was clotted by thrombin and the squeezed clot incubated in 0,017 M acetic acid, most of the activity could be recovered. This demonstrates that VA and intrinsic activator (s)(IA) bind to clots. 125I-fibrinogen labelled plasma and whole blood clots, obtained after DDAVP infusion, when incubated for 3 h at 37 °C in DDAVP serum showed 0% lysis, in normal plasma (containing weak IA activity) 15%, and in DDAVP-plasma 60% lysis. 30% lysis occurred when normal clots ware incubated in DDAVP-plasma. When DDAVP-plasma was clotted with thrombin (10 U/ml) in the presence of 0 to 20 mM Ca++, a striking inverse correlation (r=0,96) was observed between Ca++ concentration and lysis rate. The inhibitory effect of Ca++ may be due to cross-linkage of fibrin or to ↑ binding of α2-antiplasmin to fibrin.

These experiments demonstrate that physiological cone, of IA and of VA, bound to the clot during coagulation, do not induce efficient fibrinolysis in the presence of physiological Ca++ cone. Lysis takes place only when additional VA is present in the surrounding medium. This mechanism may be of physiological importance for the hemostasis-fibrinolysis balance in preventing premature lysis of formed fibrin, but assuring gradual fibrinolysis in the presence of ongoing release of VA fran vessel wall endothelium.
Antithrombotic Properties Of A Low Molecular Weight Heparin
Authors: Holmer E , Mattsson C , Nilsson S , Söderström G , Svahn C-M
Published: July 2018

The ability of heparin to prevent thrombosis has generally been assumed to be reflected by its anti-coagulant activity as measured with clotting assays such as the APTT. Recent clinical studies, however, using a low molecular weight heparin analogue, suggested that this is not necessarily always the case. The analogue had antithrombotic effect although the clotting time was only slightly affected. The present study was undertaken in order to further test this possibility in experimental animals using a low molecular weight heparin fragment with a high relative potency for inhibition of factor Xa. The heparin fragment was obtained after chemical degradation and chromatography on a antithrombin III gel. The molecular weight was 3000-4000 and the specific activity 150 U/mg and 20 U/mg as measured with an anti-Xa based assay and APTT, respectively. The fragment was tested and compared with normal mucosal heparin (corresponding activities 160 U/mg and 165 U/mg) in a rabbit thrombus model as described by Wessler. The method is based on intravenous injection of glas-activated human plasma which induces a transient hypercoagulable state during which a massive thrombus develops in stagnant blood. Thrombus formation were initiated 15 or 30 min after intravenous injection of the different heparins. Two doses, 30 and 60 U/kg (anti-Xa) of both the fragment and ordinary heparin were tested, and the results showed that the fragment was equipotent to heparin in preventing thrombus formation. However, the clotting time as measured with APTT was not significantly prolonged in rabbits treated with the fragment while a two-four fold prolongation was obtained in rabbits treated with ordinary heparin.
Anticoagulant Effect Of A New Potent Heparin In Human After Intravenous Application And The Comparison With Commercially Available Heparin
Authors: Bhargava A S , Wendt H , Günzel P
Published: July 2018

The anticoagulant effect of a new potent heparin containing 83% high affinity and 17% low affinity fractions was compared with commercial heparin after a single i.v. application in human subjects. 20 male and 20 female normal adult subjects participated in this study. 2 male and 2 female subjects per group were treated intravenously with doses ranging from 18.3–218.3 μg for new potent heparin and 40.3–483.0 μg for commercial heparin per kg body weight. Determinations of thrombin time, whole blood clotting time, activated partial thromboplastin time and plasma heparin levels using factor Xa inactivation assay were performed before and 5, 10, 15, 30 and 60 min after heparin application. The regression analysis and parallel line assay were performed using log dose or double log transformation or log dose and square root transformation of areas under the curve to estimate the relative potencies of the new heparin preparation.

The new heparin was 1.5 to 2.0 times more effective than commercial heparin per mg dry weight depending upon the coagulation tests used and the length of observation period. These relative potencies of the new heparin in human is in good correlation with relative potencies determined earlier for the same preparation in different in vitro tests including thrombelastography and in vivo using the dog as an experimental animal. In addition, the determinations of biochemical and the haematological parameters, 24h after application indicated no signs of any adverse effect.
Comparison Between The Action Of Heparin And Of Low Molecular Heparin Analogues On The Hemostatic Mechanism
Authors: Vinazzer H , Stemberger A , Haas S
Published: July 2018

Low molecular weight polysulfated polysaccharides have a considerably weaker influence on overall coagulation than heparin. The activated PTT is however,similarly prolonged by heparin and by low molecular weight heparin analogues. The substances SSHA (Luitpoldwerk, Munich, FRG) and Polyanion SP 54 (Bene-Chemie, Munich, FRG) were examined in detail and were compared to heparin. In vitro,the thrombin time was considerably prolonged by heparin but was minimally influenced by both test substances. The aPTT however, was similarly prolonged by heparin and by the test substances. A series of experiments on factor Xa resulted in a similarly strong inhibition of this activity by heparin and by the test substances whilst inactivation of thrombin was considerably stronger when heparin was present in the system. Single subcutaneous injections of 50 mg heparin given to 10 volunteers resulted in a distinct prolongation of the r- and k-time of the thrombelastogram,of the thrombin time and of the aPTT. 50 mg of the test substances given to the same group of volunteers also prolonged the aPTT but had no influence on the other parameters. The endogenous and exogenous activation of factor Xa from plasma was similarly inhibited by heparin arid by the test substances. Heparin also increased platelet aggregation by collagen. This effect was considerably weaker after injection of the low molecular weight substances. After single injections,all effects subsided within 8 hours. The minimal antithrombin enhancing potency of the two test substances causes a considerably weaker effect on overall coagulation than heparin whilst the potentiating effect of Xa-inhibition is similar in heparin and in both low molecular weight compounds.
Synthesis And Catabolism Of Human Prothrombin And Pivka-II
Authors: Tsukada H , Takada M R , Tanaka H , Takeuchi T , Gonmori H , Kobayashi N , Maekawa T
Published: July 2018

The synthesis and catabolism of both prothrombin and PIVKA-II were studied in patients with stable stage of various thromboembolic disorders. Laboratory examinations revealed that their hemostatic and fibrinolytic activities were within normal range. Thirty mg of warfarin was administered orally to patients, which was followed by daily U to 8 mg of warfarin to maintain one stage prothrombin time in therapeutic range. Fifty mg of vitamin K was administered intravenously to these anticoagulated patients. PIVKA-II was determined as prothrombin related antigen in the BaSO^ adsorbed plasma by the method of Ganrot using anti-human prothrombin antibody.

After the administration of 30 mg of warfarin, prothrombin clotting activity(FII:C) decreased exponentialy with the half life(T 1/2) of 74 hrs on an average. Rapid increase of PIVKA-II was observed in the early phase of warfarin administration, though in the later phase an increasing tendency slowed down. The changes of PIVKA-II in the early phase could be analyzed easier by following up the changes in the value of (100 - PIVKA-II activity)%. As the progress of time after the administration of warfarin, their values decreased exponentialy with the T 1/2 of 64 hrs on an average. After the administration of vitamin K, FII:C increased rapidly in the initial phase, then slowly returned to normal level. Similarly to the case of PIVKA-II after the administration of warfarin , values of (100 - FII:C activity)% decreased exponentialy with the T 1/2 of 45 hrs on an average. PIVKA-II decreased rapidly after the administration of vitamin K, describing exponential curve with the T 1/2 of 40 hrs on an average.

From the results described above, it is suggested that the catabolism of PIVKA-II is much more faster than that of FII:C.
Vitamin K Dependent Carboxylaticn Of Peptide Bound Glutamic Acid Residues
Authors: Kosow D P , Esnouf M P , Gainey A I , Hill H A O , Thornally P J
Published: July 2018

The chemical mechanism by which vitamin K promotes the posttranslational carboxylation of specific glutamic acid residues in the N-terminal region of prothrombin has not yet been elucidated. We have previously suggested that vitamin K reacts with dioxygen and carbon dioxide to form a species of active carbon. In this study we have investigated the reaction of reduced vitamin K in alcoholic solution with dioxygen in the presence and absence of carbon dioxide. We find that carbon dioxide is necessary for the rapid formation of vitamin K oxide. Vitamin K oxide was formed when either cis or trans vitamin K was used. However, trans vitamin K is specifically required in enzymatic carboxylation studies. We propose that in rat liver microsomal preparations the carboxylation of synthetic peptide substrates is coupled to the chemical epoxidation of vitamin K by the carboxylase.
Prothrombin And Abnormal (Des-γ-Carboxy) Prothrombin: Assessment Of Plasma Levels By Ria In Disorders Of Hepatic Vitamin K-Dependent Carboxylation
Authors: Blanchard R A , Furie B C , Kruger S F , Furie B
Published: July 2018

The vitamin K-dependent blood coagulation zymogens contain γ-carboxyglutamic acid (Gla) in their amino terminal domains. The presence of these Gla residues is necessary for the physiologic activation of these zymogens to proteases. The Gla residues are synthesized from glutamic acids on precursor proteins in the liver by a vitamin K-dependent carboxylase. In treatment with vitamin K antagonists or in the presence of vitamin K-deficiency carboxylation is impaired and des-γ-carboxy forms circulate in the blood. We have developed specific RIAs for human prothrombin (NPT) and for des-γ-carboxy human prothrombin (APT) to evaluate the levels of these species in human plasma. These plasma assays measure only NPT despite the presence of APT or only APT despite the presence of NPT.

These results suggest that total prothrombin synthesis is decreased in patients treated with warfarin and in patients with intrinsic liver disease compared to normals or to vitamin K-deficient patients. APT is not a component of normal plasma (>0.03 g/ml). However APT is present in 90% of the patients with intrinsic liver disease. In ten patients with liver disease in whom samples were obtained before and after vitamin K administration APT persisted in the plasma. This indicates impaired vitamin-K-dependent car-boxylation in these disorders. We feel that the patterns of plasma NPT and APT in these disease states are characteristic and may prove useful in identifying and distinguishing liver disease from disorders of PT biosynthesis.
Cleavage Of Human Prothrombin By Partially Purified Echis Carinatus Venom
Authors: Briét E , Griffith M J , Soule P B , Jordan S H , Braunstein K M , Roberts H R
Published: July 2018

A number of studies have been reported in which the procoagulant activity of Echis carinatus venom (ECV) has been used to investigate the structure-function of human and bovine prothrombin (II). While it is clear that ECV activates II by cleaving between the A chain-B-chain of the thrombin region of the zymogen,, the literature is not clear on the activity of ECV in terms of cleaving at other sites. Specifically, one report suggested that ECV cleaves II between the Fragment 1 and prethrombin 1 regions (F 1-Pre 1) of the zymogen and that this cleavage is required for further cleavage and activation of II. Another report suggested that ECV does not cleave the F 1-Pre 1 bond at all. The ambiguity has arisen because of the well-known sensitivity of the F 1-Pre 1 bond to cleavage by thrombin and the inability of most thrombin inhibitors to block thrombin activity during the course of II activation by ECV. To this point, diisopropylphosphofluoridate, isoleucyl-pro-pyl-arginine chloromethyl ketone, and benzamidine did not prevent significant F 1-Pre 1 cleavage by ECV in the present study, Dansyl-arginine-N-(3-ethyl-l, 5-pentanediyl amide) (DAPA) however, appeared to afford nearly complete protection against F 1-Pre 1 cleavage during ECV activation of II. Since some F 1-Pre 1 cleavage was observed even in the presence of 10-3M DAPA (Ki = 10-7M) it appeared that ECV might cleave this bond at a relatively slow rate. This was confirmed by the results of experiments in which the rate of F 1-Pre 1 cleavage in the presence of DAPA was found to be proportional to the concentration of ECV. In addition, EDTA, which inhibits ECV activity, blocked further F 1-Pre 1 cleavage at a time when the conversion of prothrombin to meizothrombin was complete. Human prothrombin appeared to be much more susceptible to F 1-Pre 1 cleavage by ECV than bovine prothrombin. Our results suggest that the activation of prothrombin by ECV does not require F 1-Pre 1 cleavage by ECV as suggested by other investigators.
Feiba And Autoplex: A Comparison Of These Two Activated Prothrombin Complex Concentrates
Authors: Lechler E , Eggeling B , Meyer-Börnecke D , Stoy H
Published: July 2018

These activated concentrates are used for the treatment of patients with factor VIII inhibitors . Both shorten the activated and non-activated partial thromboplastin time of inhibitor plasma and hemophilia A plasma in vitro. They do not or only to a minor degree improve the prothrombin consumption of hemophilia A plasma in vitro. In gel filtration of AUTOPLEX the activity which shortens the PTT of hemophilia A plasma eluted in a volume higher than that of the nonactivated factors of the prothrombin complex and contains activated factor IX. The activity of FEIBA elutes at a lower filtration volume in a rather broad peak together with the factors of the the non-activated prothrombin complex. BaSO4- adsorbed plasma and purified antithrombin (Behring) abolish the activity of AUTOPLEX more readily than of FEIBA. Both concentrates have only a low amidolytic effect (S 2222) and are not inhibited with SBTI and PMSF. In the crossed two-dimensional immunelectrophoresis with heparin in the agarose of the first dimension and anti-antithrombin (Behring) in the agarose of the second dimension (method of Sas), a mixture of AUTOPLEX and antithrombin results into a two peak precipitation of antithrombin, whereas with FEIBA a broadened intermediate peak develops. In vivo both concentrates do not improve the prothrombin consumption and AUTOPLEX shortens the PTT for at least 90 minutes. In summary, these two concentrates differ considerably.
Dosage-Efficacy Relationship In Single-Dose Replacement Therapy For Acute Bleeding Episodes In Hemophilia
Authors: Weiss A E
Published: July 2018

The cardinal principle of modern therapy for acute bleeding episodes in hemophilia is “early adequate treatment.” To better define “adequate”, the dose-efficacy relationship was studied in treatment of 491 acute hem- arthroses in 14 adolescent and adult severe hemophiliacs by prompt factor concentrate infusion. All treatments were given within 5 hrs after first recognition of bleeding, with the mean delay being 1.9 hrs; 68% were given within 2 hrs, and 86% within 3 hrs. Treatments were given in 3 concentrate dosage ranges: 8-11, 11-15, and 15-18 u/kg. The outcome of treatment was graded as a “success” (S) if bleeding symptoms resolved promptly without need for additional infusions or absence from school or work; “marginal” (M) if treatment was otherwise successful but rebleeding occurred at the same site within 10 days; or “failure” (F) if symptoms persisted and required additional infusions and/or absenteeism. In each dosage range the time from bleed recognition until treatment and the concentrate dosage were not significantly different among the outcome groups. However, there was a highly significant difference (p<0.01) in the efficacy of treatment at the 3 dosage ranges (see table). The success rate increased stepwise, and the failure rate decreased with increasing dosage ranges. Similarly, the frequency of rebleeding decreased with increasing dosage for primary treatment. The 10% failure rate with low doses is in agreement with several reports advocating low doses for early treatment, but the higher success rates and lower failure and rebleeding rates with the higher doses indicate that these parameters and their impact on overall bleeding frequency and concentrate usage must be considered in the determination of “adequate” doses.
Measurement Of Inhibitors To Procoagulant Factor VIII (VIIIC) By Immunoradiometric Assay (IRMA)
Authors: Furlong R A , Peake I R , Bloom A L
Published: July 2018

Antibodies against VIIIC (VIIICAg) were assayed using a modification of a two-site solid phase IRMA for factor VIII clotting antigen (VIIICAg). Anti-VIIICAg antibodies obtained from a multi- transfused haemophiliac were separated as IgG and labelled with I125. This was used to test plasma from patients with factor VIII inhibitor by competetive binding to common antigenic sites on immunoimobilised VIIIC. A haemophilic inhibitor assessed as 225u by the Bethesda method was used as standard. Results of inhibitor assay using the IRMA in 19 plasma samples from 15 severe haemophiliacs were similar to those obtained by the coagulation method. The increased sensitivity by IRMA of 0.01 u/ml enabled measurement of a haemophilic inhibitor undetectable by clotting assay. Anti VIIICAg activity was also detectable in plasmas from three individuals with acquired inhibitors against VIIIC. These plasmas which also had measurable residual VIIICAg gave dilution curves non-parallel to the standard haemophilic plasma curve. Measurement of haemophilic inhibitors using three IRMAs each employing different I125 labelled haemophilic anti VIIICAg antibodies showed that there was no difference in the sensitivity of the three assays but in some plasmas the results were discrepant indicating different specificities of the labelled antibodies.
Evidence Of Possible Linkage Between Von Willebrand’S Disease (VWD) And 2 Polymorphic Genetic Markers
Authors: Barrow E S , Reisner H M , Namboodiri K K , Elston R C , Graham J B
Published: July 2018

VWD shows great variability within and between families. In some kindred the affected persons show decreased amounts of an apparently normal VIIIR:Ag. In others, abnormal electrophoretic mobility of VIIIR:Ag has been associated with a defect in the degree of polymerization. Within kindred, expressivity may be so variable that some transmitters have normal laboratory findings. Detection of linkage between VWD and a clearly defined antigenic or biochemical marker might provide the means to make unambiguous diagnoses and to distinguish between the effects of the multiple genes involved in synthesis of VIIIR:Ag.

We have examined 4 VWD kindred, using 23 genetic markers. Individuals were classified for VWD using clinical and laboratory data, pedigree information and 2 statistical procedures: D I based on 3 measures of F VIII activity, and D II which also included bleeding time and a subjective index of symptoms. Using D I, a LOD score of 0.66 at a recombination frequency (θ) of 0.20 was found with GPT (glucose pyruvic transaminase). Using D II a LOD score of 0.50 at a θ of 0.25 was found. Most of the evidence of a VWD- GPT linkage was provided by a single kindred. Using D II, evidence suggesting a second linkage was observed between VWD and GLO (glyoxylase) with a LOD score of 1.03 at a 0 of 0.20, all 4 families contributing.

The LOD scores reported are suggestive of linkage and warrant further study. Since GLO relates to Chromosome 6 while the chromosomal location of GPT is not known and since GPT and GLO are unlinked, VWD may be genetically heterogeneous, 2 or more loci being involved.

Studies to clarify the relationship of VWD to Chromosome 6 are underway using markers known to be linked to GLO.
In Vitro Bleeding Time
Authors: Sawada S , Kimura A , Yoshida Y
Published: July 2018

One of the authors, A.Kimura,et al. reported the technique called “in vitro bleeding time” using fresh pig skin and the heparinized animal blood (BLOOD 54:6 1347-1357). The system was used to measure both the normal hemostasis of swine and to investigate what causes abnormal hemostasis in the von Willebrand animals. Author modified this system to use dried pig skin and heparinized normal(n=62) and abnormal hemostatic human blood(n=20). Heparinized human whole blood at constant hydrostatic pressure was allowed to flow through a 5mm incision in the dried pig skin (LYODERM, ARMOUR PHERMACEUTICAL CO.) in the flow cell(HELLI- GE CO.), shown in the figure. Exuded blood was drawn by the proportionning pump and hemolized with distilled water. The hemoglobin concentration was measured and recorded by the automatic electrophotometer. The time elapsed from the start of flow to the cessation was measured as in vitro bleeding time. In vitro bleeding time was 7.6±3.6(mean±SD) minutes in normal.

In von Willebrand’s disease and Hermansky-Pudlak syndrome in vitro bleeding time was prolonged.
Platelet Volume Measurements: The Importance Of Anticoagulation
Authors: Thompson C B , Diaz D D , Quinn P G , Lapins M , Kurtz S B , Valeri C R
Published: July 2018

Recent advances in electrical cell sizing have made mean platelet volume (MPV) routinely available in most clinical laboratories. To study the importance of anticoagulation on platelet size stability, blood was collected in 7 different anticoagulants and stored at room temperature for up to 8 hours. Platelet counts and platelet sizing were performed using whole blood on a Coulter S+ and using platelet-rich plasma on a Coulter H4 Channelyzer. The results suggest that both calcium chelation and acidification were required to inhibit platelet shape change and aggregation. Electrolyte composition, pH, and tonicity of the anticoagulant all influenced the stability of the MPV. As a result of these studies, an anticoagulant combining ACD and Na2EDTA at a pH of 5.0 and an osmotic strength of 308 m0sm/1 was used to study platelet volume and counts in whole blood on a Coulter S+ used in the hematology laboratory of our hospital. Platelet counts with ACD-Na2EDTA anticoagulant were no different from routine Na2EDTA platelet counts and exhibited 2.9% error in reproducibility and a 4.4% variability over the 8 hours of storage. Mean platelet volumes were reproducible to within 3% and had less than 1% variability over the 8 hours of storage. Platelets anticoagulated with ACD-Na2EDTA remained discoid in shape and could be shown to undergo a shape change on stimulation with ADP up to at least 8 hours after collection, when the pH was adjusted to 7.4 and the Ca++ concentration restored.

These data demonstrate that platelet count and platelet volumes remained stable in blood collected in ACD-Na2EDTA anticoagulant for up to 8 hours at room temperature. In 52 volunteers studied, an inverse correlation (r=.72, p<0.001) was observed between platelet count and MPV, suggesting that the circulating platelet mass may be a more important indicator of platelet homeostasis than either the platelet count or the mean platelet volume alone.
A Platelet Aggregometer With Automatic Data Processing
Authors: Blasberg P , Wurzinger L J , Mussler K , Myrenne H , Schmid-Schönbein H
Published: July 2018

A new aggregometer was designed to meet the following requirements: A) a uniform well defined shear rate throughout the whole PRP sample, B) two different shear rates - one favourable for platelet aggregation (PA) - one to subject already formed aggregates to high shear to test aggregate stability, C) a modular set up to extend the aggregometer easily from 1 to 4 channels, D) automatic processing and documentation of all aggregation data.

I. The aggregation moduls: Each aggregation modul contains a Couette-viscometer (500 μl PRP required). The outer cylinder (a disposable plastic vial) is rotated to induce shear rates in the range of 40s-1 and 4000s-1. For measurement of PA each channel is equipped with a He-Ne laser which radiates a light beam through the sample. Transmitted (It) and scattered (Is) light is measured by independent photodiodes and from the ratio Is/It+Is turbidity of the test sample is estimated. The moduls are thermostatised to 37°C. 1 to 4 moduls are supplied by one motor-gear box (1:100) unit. 2. The data processing unit: The data processing unit is controlled by an OEM-microcomputer, which allows an on line display and storage of the actual turbidity values during the whole experiment. At the end of each experiment the aggregation curve is calculated and may be plotted on a x,t- recorder. The area under the curve and the lag phase of PA is estimated. In addition the whole experimental run is controlled and documented by the printer equipped microcomputer using the interactive high level language BASIC.

The aggregometer consists mainly of two parts:
The described aggregometer exhibits the following advantages: the use of a uniform low shear rate enables one to observe spontaneous PA and to reduce the concentrations of PA-inducing agents (e.g. 5×10-7m ADP) compared to conventional aggregometers whereas the stability of already formed aggregates can be tested with the high shear rate. Furthermore the evaluation of all data (PA, integral of PA, lag time) is done automatically.
Mechanism Of Regulation Of Platelet Amp Deaminase
Authors: Ashby B , Holmsen H
Published: July 2018

Metabolic ATP is converted rapidly to IMP during platelet secretion. The conversion is rate limited by deamination of AMP to IMP. AMP deaminase has been purified to homogeneity from human platelet concentrates by phosphocellulose chromatography. Kinetic studies showed sigmoidal behavior as a function of AMP with K0.5 values of 3.5 and 4.0 mM in 0.1 M NaCl and 0.1 M KC1, respectively, at pH 6.5. Activation by saturating ATP converted the velocity versus substrate plot to hyperbolic with a Km of 1.2 mM in either salt. Addition of increasing concentrations of GTP in the presence of NaCl led to activation followed by weak competitive inhibition whereas GTP in the presence.of KC1 gave strong competitive inhibition with no apparent activation. Consequently, enzyme activity measured at 100 μM AMP in the presence of 10 μM GTP was 22 times greater in NaCl than in KC1. Studies on the effects of pH revealed pH optima around 6.5 in NaCl and 6.7 in KC1. Activation by ATP or GTP shifted the optima to lower pH values whereas inhibition by GTP shifted the optima to higher pH values. All of these results may be described by a model that does not invoke subunit-subunit interactions to explain cooperative behavior but requires obligatory binding of AMP to a distinct activator site on the same subunit before AMP can bind to the catalytic site. ATP and GTP bind to the same activator site as AMP and GTP can also bind competitively to the catalytic site. Changes in activity produced by NaCl or KC1 or pH are due to changes in the affinity of the two sites for the various nucleotides in media of different ionic composition and pH. Regulation of AMP deaminase in the intact platelet is thus envisaged as being the result of changes in ionic composition and pH that may accompany platelet stimulation rather than changes in the concentration of effector nucleotides.
Snake Venom Phospholipases A2And Blood Coagulation : Competition Between Phospholipases And Blood Clotting Factors U And U At The Phospholipid Interface
Authors: Prigentdachary J , Boisseau M R , Dufourcq J
Published: July 2018

In a preceeding paper (J. Biol. Chem., 255, 7734-7739, 1980) we have shown that phospholipases A2 from snake venoms have different phospholipid binding affinities according, to their anticoagulant activities : only the anticoagulant one are able to bind with neutral and negative phospholipid bilayers in EDTA and therefore in coagulation test could hydrolyse both classes of lipids. A step forward in the understanding of the mechanism of the inhibitory action of phospholipases on coagulation is to know if competition between clotting factors and phospholipase can occur at the plasma phospholipid interface as a consequence of the strong affinity of anticoagulant phospholipase for lipids. The method used is mainly to look at the intrinsic fluorescence of lipids bound phospholipase when blood clotting factors II and IX are added to the complex. Results show that both factors can expel the phospholipase from the interface when the factor to phospholipase molar ratio is about 1, suggesting a better affinity of the clotting proteins for the interface. When hydrolysis of phospholipids is allowed to occur (ie in presence of calcium ion) factors also have a good affinity for degraded products. By the same technic, it was also possible to dectect direct interactions between phospholipase and factors, which differ for factors II and factor IX. This can also occur in presence of phospholipids. In summary, as it was demonstrated that inactivated phospholipase has lost its anticoagulant activity lipid hydrolysis is a necessary step. However, we show here that competition between clotting factors and phospholipase could also occur if drastic concentrations of phospholipase are used in coagulation test, i.e. molar ratio much higher than 1. It is also proposed that factor to phospholipase binding may be important in the inhibitory mechanism since clotting proteins could be no longer available for the coagulation.
Immunochemical Characterisation Of Two Human Factor IX Inhibitors And Treatment With Activated Concentrate
Authors: Giddings J C , Bloom A L
Published: July 2018

Plasma was obtained from two unrelated patients (A and B) with severe hereditary factor IX deficiency who had developed specific inhibitors of factor IX. Immunoglobulin (Ig) was fractionated by salt precipitation and ion exchange chromatography and resulted in 90% recovery of the factor IX inhibitor. Inhibitor plasma or Ig fractions were mixed with specific antisera to ig chains, incubated and centrifuged. Supernatant was assayed for residual inhibitor. Antisera, specific for the heavy chains of human IgG, IgA, IgM, IgD and IgE and the subclasses 1 to 4 of human IgG were used together with antisera specific for the kappa and lambda light chains of IgG. Results of inhibitor neutralisation assays were the same whether plasma or Ig fractions were used. Inhibitor from patient A was completely neutralised by anti IgG and by anti-lambda serum, and was largely neutralised by antiserum to IgG subclass 4. Other antisera had no effect. Inhibitor from patient B was completely neutralised by anti IgG and partially neutralised by anti-kappa and anti-lambda serum. Partial neutralisation of inhibitor was also seen with antisera to IgG subclass 1 and IgG subclass 4. Other antisera again had no effect. Inhibitor A inactivated factor IX instantaneously to a titre of 1 in 100. Neither conventional factor IX concentrate (Oxford DE) at 1 u/ml nor activated‘factor IX’ concentrate (Feiba, Immuno) at 1 u/ml (‘activity’ unit) corrected the kaolin cephalin clotting time (KCCT) of patient A in vitro. However after intravenous administration of Feiba (25 u/kg) progressive shortening of the KCCT occurred from 127 sec. to 60 sec. at 60 min. and was followed by rapid resolution of a haemarthrosis. It is concluded that factor IX antibodies may be of monoclonal or oligoclonal immunoglobulin class and that on development, therapeutic trial of activated factor IX-prothrombin concentrates is indicated for the control of bleeding.
Use Of Poly(L-Lysine)-Sepharose 4B For Isolation Of Vitamin K Dependent Factors From Human Plasma
Authors: Mohammad S F , Martin B , Chuang H Y K , Sharma N C , Mason R G
Published: July 2018

Poly(L-lysine) (PLL; Mr=15,000-40,000) or human albumin was conjugated with Sepharose 4B-CNBr to obtain PLL-Sepha- rose 4B or albumin Sepharose 4B. When human citrated plasma was passed through a column (3×1 cm) of PLL-Sepharose 4B, it was observed that eluted plasma had a prolonged PTT time and further analysis showed that it was completely deficient in factors II, VII, IX, and X. Some loss of factors V, VIII, XI and XII was also noticed. Plasma passed through a column of albumin under identical conditions suffered no detectable loss of factors II, VII, IX and X. Loss of factors V, VIII, XI and XII was also reduced. Factors II, VII, IX and X that adsorbed to PLL-Sepharose 4B column did not elute from the column even after extensive washing (100 ml of 0.15 M NaCl containing 0.018 M sodium citrate in 5 ml aliquots). Immunoelectrophoretic examination of the eluate showed that it was essentially free of albumin indicating that the washing of the gel was satisfactory. Also, no other protein was detectable in the wash. Addition of 0.75 M NaCl containing 0.018 M sodium citrate to the washed gel resulted in the elution of factors II, VII, IX and X. Addition of the high salt eluate (after adjustment of ionic strength to 0.15 M NaCl with distilled water) to the plasma previously made deficient by passage through the PLL-Sepharose 4B column resulted in normalization of the PTT time. Subsequent analysis of the high salt eluate revealed that the recovery of various factors was: II=88%, VII=81%, IX=79% anl X=92%. Preliminary studies indicated also that the eluate from PLL-Sepharose 4B was apparently devoid of activated factor X. The possibility of other activated blood clotting factors being present in the PLL-Sepharose 4B eluate is currently under investigation.
Semi-Automation Of Cryoprecipited Factor VIII Production
Authors: Wensley R , Fletcher S , Snape T
Published: July 2018

The essential processing stages of cryoprecipitate production include plasma-separation, freezing, thawing and harvesting. An automatic cabinet has been developed to perform the freezing and thawing stages according to a predetermined time sequence. Employing conventional refrigeration and operating over a temperature range of −70°C to ?30°C, a full cabinet load of 90 bags can be frozen in 30 minutes and thawed in 90 minutes while being rotated in the high air flow. Use of the freeze-thaw cabinet has resulted both in high yield and reproducibility of cryoprecipitates and has permitted increased cryoprecipitate production with energy saving sufficient to offset the capital cost of the equipment.
The Natural History Of Hemophilic Factor VIII-Antibodies: Differences In The Half Disappearance Time Following Anamnestic Rise
Authors: Lechner K , Krinninger B , Niessner H , Nowotny Ch , Thaler E , Deutsch E
Published: July 2018

In 12 severe hemophiliacs with an antibody to factor VIII of the high responder type antibody activity was followed over a treatment free period of at least three months up to seven years, following 30 factor VIII treatments. After reaching the peak level the antibody titer declined exponentially with a median half disappearance time of 36 days within in a period of 2 - 7 months. There was a considerable variation of the half disappearance time among patients and even in the same patient. Particularly inter - esting were two patients who showed an unusually short half disappearance time of 5 and 9,5 days,respectively. On the other hand, in one patient the half disappearance time after three stimulations was between 67 and 98 days.

In five patients a biexponential disappearance curve was observed. In these patients after a first component of rapid decline (as described above) a second slower component was observed. The half disappearance time of this second component was between 92 - 480 days (median 200 days). In one patient eventually a plateau was reached. The transition of the first to the second component occured 2-7 months after stimulation, when the antibody level had fallen to 2 - 30 % of the peak level. Biexponential curves were observed only in patients with peak levels of more than 300 BU/ml. However, only a single rapid component was observed in one patient with peak levels of 470 BU/ml. Hemophilic factor VIII antibodies are heterogenous with regard to their behaviour in vivo. These differences have to be taken into account if therapeutic decisions have to be made.
An Inhibitor Selectively Directed Against Factor VIII-AHF In A Patient With Von Willebrand’S Disease (vWD)
Authors: Castella A , Miller J L , Neuberg R W , Friedman H S
Published: July 2018

We have studied a 17 month old boy with previously diagnosed vWD who presented with intracerebral hemorrhage. There was a history of excessive bleeding from circumcision, after corrective surgery for pyloric stenosis, and from an upper lip cut. He had received multiple units of cryoprecipitate from the first month of life until the present admission. Work-up demonstrated prolonged bleeding time and prolonged PTT, normal prothrombin time, thrombin time, and platelet aggregation with ADP, epinephrine and collagen. Ten days after cessation of factor VIII replacement therapy he had undetectable F VIII-AHF, F VIII-Ag of 86% by the Laurell technique and F VIII-RCo of 18%. We could demonstrate a time- dependent inhibitor directed against F VIII-AHF measuring 92 Bethesda units. In mixing studies with normal plasma, little or nor inhibitory activity directed against F VIII-RCo could be identified. The patient’s platelets showed increased ristocetin-induced platelet aggregation (RIPA) at doses as low as 0.5 mg/ml.

There was no evidence of a bleeding disorder in the maternal side of the family. All of the paternal family members studied showed prolonged bleeding times, decreased F VIII-RCo, and increased RIPA with low doses of ristocetin, findings similar to those patients recently characterized as having type IIb vWD. Although other members of the patient’s family had previously received cryoprecipitate replacement therapy, no inhibitors appear to have developed in these persons.

Factor VIII inhibitors in vWD are uncommon, with those reported usually showing little or no inhibition of F VIII-AHF. These findings appear to represent a unique pattern of inhibitor development that may be related to a molecular variant of vWD in this patient.
An IGM Circulating Anticoagulant Associated With Small Cell Carcinoma Of The Lung
Authors: Handorf C R , Ricardo M J , White F L
Published: July 2018

A 62 year old white female with small cell carcinoma of the lung presented with a prolonged activated partial thromboplastin time (APTT). The patient had no history of abnormal bleeding but previously had pulmonary emboli. Coagulation parameters other than APTT were within normal limits. Pertinent laboratory data included an IgM lambda paraprotein. Dilution studies indicated the presence of a high titer circulating anticoagulant; specific assay revealed marked inhibition of factor VIII (50 Bethesda units). Factor VIII activity was 26% of normal. A purified IgM fraction of the patient’s plasma was obtained by filtration on a Bio-Gel A-1.5m column. When incubated with normal plasma the IgM fraction demonstrated anticoagulant activity by prolonging the APTT.

The patient underwent plasmapheresis with albumin and plasma exchange which significantly corrected the APTT. The combination of small cell carcinoma of the lung and IgM paraprotein with anti-factor VIII activity represents an unusual paraneoplastic syndrome; to our knowledge this combination has not been reported previously.
Study On The Probability Of Carriership In Hemophilia A
Authors: Blanco A N , Elgue G E , Canaveri A A Martinez , Novaro S
Published: July 2018

The aim of this study was to find a reliable method to detect carriers of hemophilia A. Eighteen obligatory carriers, 14 possible carriers, without a family history but with hemophilic son, and 29 normals were studied. The three groups were in the same age range (19 to 47) had no intake of oral contraceptives and the blood samples were taken in the middle of the menstrual cycle. Factor VIII activity (Factor VIII:C) was determined by the one stage method and the Factor VIII-related antigen (Factor VIIIR:Ag) by Laurell’s immunoelectrophoresis.

In the first group we confirmed the carrier state in 72.2% (13/18) by discriminant analysis and 88.9% (16/18) on the basis of the ratio of Factor VIII:C to Factor VIIIR:Ag or its logarithmic transformation. In our Laboratory the logarithmic transformation of the ratio showed the least percentage of misclassification so it proved to be more efficient.

To arrive to the final statement concerning the probability of being a carrier we combined laboratory data and pedigree information. In 64.3% (9/14) of the possible carriers the analysis of the laboratory data (by logarithmic transformation) increased their final probability of being carriers.