bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Weight Determinations Of Low Molecular Weight (LMW) Heparins By Ultracentrifugation And High Pressure Liquid Chromatography
Authors: Barlow Grant , Sugisaka N , Petracek F J
Published: July 2018

Molecular weights were independently determined on nitrous acid depolymerized LMW heparin fractions ranging from 2-15 daltons using the analytical ultracentrifuge and high pressure liquid chromatography (HPLC).

Sedimentation-diffusion equilibria were obtained in the analytical ultracentrifuge using speeds ranging from 20,000 to 56,000 rpm. Near theta conditions were obtained using 0.5M NaCl as the solvent. Calculations of molecular weight distributions and, from those figures, weight average molecular weights were made using the method described by Scholte (N.Y. Acad Sci. 164, 156, 1969). The results show that weight average values as low as 2,000 daltons can be determined.

Sedimentation-diffusion equilibria were obtained in the analytical ultracentrifuge using speeds ranging from 20,000 to 56,000 rpm. Near theta conditions were obtained using 0.5M NaCl as the solvent. Calculations of molecular weight distributions and, from those figures, weight average molecular weights were made using the method described by Scholte (N.Y. Acad Sci. 164, 156, 1969). The results show that weight average values as low as 2,000 daltons can be determined.

The HPLC results were obtained using previously described methods (Fed Proc. 36, 89, 1977) and a new highly efficient gel column (TSK gels). Fractionated dextrans were used as reference standards.
Significance Of Tryptophan Residues In Fibrinogen In Fibrin Polymer Formation
Authors: Matsushima A , Saito Y , Okano K , Inada Y
Published: July 2018

Human fibrinogen and its fragment were modified with 2-hydroxy-5-nitrobenzyl bromide(HNBB) or with H2O2-dioxane to see the functional significance of tryptophan residues in the fibrinogen molecule. Tryptophan residues were modified with HNBB in a stepwise manner. Modification of two out of the total 64 tryptophan residues in the molecule did not reduce the polymerization activity with thrombin but further modification of the next two residues led to a complete loss of the polymerization activity. The latter two tryptophan residues modified were in Fragment E and the position may be either Trp 33 or Trp 41 in the a-chain in the fibrinogen molecule.

Fragment D inhibited the association activity of fibrin monomer. This inhibitory effect was lost completely by the modification of approximately six out of the total 21 tryptophan residues in the fragment with H2O2-dioxane. These resul ts indicate the significance of tryptophan residues in Fragments D and E of fibrinogen molecule in fibrin polymer formation.
Treatment Of Venous Thromboembolism - Case For Thrombolytic Therapy
Authors: Kakkar V V
Published: July 2018

Thrombolytic therapy has a unique advantage in the treatment of patients suffering from thrombotic disease, since it is capable of inducing the dissolution of intravascular fibrin and thus causing the reduction or elimination of thrombi. The rapidity of thrombus removal distinguishes this form of treatment from anticoagulant therapy, in which normal physiological processes are allowed to restore the obstructed circulation. By quickly removing the obstruction, it should be possible to reduce the mortality arising from acute thromboembolic episodes.

The results of therapy for deep-vein thrombosis have been fairly uniform. The published studies can be broadly classified into two main groups; in uncontrolled trials, partial or complete lysis of thrombi was obtained in approximately 65-80% of the patients who received streptokinase, while only 10-25% of the patients receiving heparin showed this change.

In patients suffering from acute major or massive pulmonary embolism, a number of trials have demonstrated a more rapid resolution of the embolus than would be expected by treatment with heparin alone.

The role of lytic therapy in preventing the late sequelae of deep vein thrombosis at present remains uncertian. Studies involving large numbers of patients and longer periods of follow-up are required to determine the extent to which post phlebitic venous insufficiency is reduced by early thrombolytic therapy.
Inhibition Of Contact Activation By Platelet Factor 4
Authors: Weerasinghe K , Scully M F , Kakkar V V
Published: July 2018

The contact activation of FXII by dextran sulphate (DS, MW 500,000) has been shown to be inhibited by platelet factor 4 (PF4). Plasma was prepared from blood taken with EDTA, theophylline and PGE1. Contact activation was initiated by the addition of an equal volume of DS (10μg/ml) and incubating at 0° (8 min) or 37° (2 min). The kallikrein generated was measured using S2302. Addition of purified PF4 to plasma caused a concentration dependent inhibition with complete inhibition at greater than 2.0μg/PF4/ml plasma (molar ratio of 20:1). PF4 was also observed in a concentration dependent manner to inhibit clotting of plasma as initiated by dextran sulphate.

Repeated freezing of human PRP (Av. platelet count=3.55 × 108/ml) released PF4 (as measured by radioimmunoassay) and caused inhibition of contact activation. By dilution with PPP, the degree of inhibition was found to be related to the concentration of PF4. The amount needed for 50% inhibition varied from 0.2-1.5μg/ml in different donors. Platelet aggregation with epinephrine (2μg/ml) released 4.8μg/ml (±1.7) of PF4 and showed 55% (±22) inhibition of contact activation (n=ll). Collagen (5.7μg/ml) caused 4.9μg/ml (±1.3) of PF4 to be released, but the degree of inhibition varied (n=13). The discrepancy between this and the freezing experiment may be due to the “contact-product activator” properties of collagen and the intact platelet membrane. Control experiments performed using indomethacin or EDTA-PRP showed no release of PF4 or inhibition of contact activation.

The effect observed appears to be related directly to the ability of PF4 to bind to negatively charged polysaccharides since PF4 was not found to inhibit activated factor XII or kallikrein. These results indicate another role for PF4 as an inhibitor of contact activation. Together with its known antiheparin properties it indicates that PF4 may be closely involved in the control of haemostasis.
Computerized Axial Tomographic (CAT) Diagnosis Of Vascular Thrombosis And Patency Of Aorto-Coronary Bypass Graft
Authors: Moncada Rogelio , Salinas Matias , Churchill Robert , Love Leon , Reynes Carlos , Fareed Jawed , Parvez Zaheer , Messmore Harry L
Published: July 2018

During the past decade, aorto-coronary bypass surgery (ACBS) has been widely prescribed as treatment of angina pectoris. Dysfunction or closure of an aortocoronary bypass graft (ACBG) is generally suggested by a number of vague subjective complaints and few, if any, objective signs derived by history, physical examination, resting and stress E.K.G., and lately by Thallium201 regional myocardial perfusion studies. Definitive confirmation of graft closure or stenosis has traditionally required post operative selective catheterization and angiography of the grafts in question. We have utilized CAT to determine patency of an ACBG with encouraging results. Biological motion has been the principle deterrent to the use of CAT in evaluating the coronary arteries and cardiac chambers. Third and fourth generation scanners have been employed to explore the feasibility and potential use of CAT in imaging cardiac structures. Surprisingly, excellent quality images are currently being obtained with exposure times of one to five seconds, even though some of these images are reconstructed from repetitive data obtained during one or more cardiac cycles partially degrading the image. CAT demonstration of the cross-sectional anatomy combined with simultaneous contrast media enhancement represents the basic ingredients utilized to demonstrate the patency of ACBG. Sixty-five patients have undergone this procedure after single or multiple revascularization procedures. Eighteen of these patients, with a total of thirty-three grafts, had correlative selective angiography of the individual venous grafts. Twenty-two grafts proved to be patent with both techniques while ten of eleven thrombosed grafts were properly predicted by CAT. The benefits of this relatively non-invasice, safe, repeatable, and accurate method for detectinq ACBG is presented.
Altered Regulation Of Prostacyclin (PGI2) Activity In Severe Pre-Eclampsia
Authors: de Gaetano G , Marchesi D , Zoja C O , Schieppati A , Misiani R , Mecca G , Rossi E , Donati M B , Remuzzi G
Published: July 2018

It has been suggested that prostaglandins of the E series play a crucial role in regulation of fetal vascular tone. The discovery of PGI2 has opened new perspectives for understanding the circulatory physiology of normal pregnancy and the vascular abnormalities of pre-eclampsia. We have bioassayed PGl2 platelet antiaggregatory activity and confirmed it by RIA of 6-Keto-PGF1α and thin-layer radiochranatograpty in human umbilical and piacental vessels. Fetal umbilical arteries obtained at the end of 9 uncomplicated pregnancies generated significantly more PGI2 (191 ± 29 ng/mg tissue) than vessels from control non pregnant adults (43 ± 12 ng/ mg) (p < 0.01). Placental veins generated PGI2 similarly to control veins (33 ± 7 and 37 ± 8 ng/mg respectively) . This could contribute to maintaining the lew peripheral vascular resistance of the fetal circulation with very high cardiac output. Umbilical arteries from 5 vramen with severe preeclampsia generated significantly less PGI2 (65 ± 12 ng/mg tissue) than the corresponding vessels from women with normal pregnancy (p < O.Ol). Similarly placental veins frem preeclampsia patients generated less PGI2 (11 ± 2) than normal pregnancy veins (p<0.01).

We propose that in severe pre-eclampsia,placental ischemia and fetal distress could be triggered by deficiency of the adaptive mechanism which maintains high vascular levels of PGI2 in normal pregnancy. The abnormally high PGI2 stirnulating activity in plasma frem patients with severe pre-eclampsia in comparison with plasma from women with normal pregnancy supports the contention that regulation of PGI2 synthesis is deranged in pre-eclampsia.
Thromboxane B2 And Betathromboglobulin In Symptomatic Coronary Artery Disease
Authors: de Boer A C , Turpie A G G , Butt R , Genton E
Published: July 2018

To investigate pattern and clinical value of platelet release and thromboxane synthesis in myocardial ischemia and necrosis, plasma (P) thromboxane B2 (TXB2) (normal 162 ± 28 pg/ml, mean ± SEM), P-betathromboglobulin (BTG)(24 ± 2 ng/ml) and urine (U) BTG (0.18 ± 0.04 ng/ml) were measured by radioimmunoassay in 98 patients admitted to the CCU with chest pain thought to be cardiac. Final diagnoses were 26 patients with non-cardiac pain (Gp I), 47 patients with angina (Gp II) and 25 patients with acute MI (Gp III). P- BTG and TXB2 were measured at presentation and on day 1, 2 and 3, U-BTG on Day 1, 2 and 3. Gp I; P-BTG (26 ± 3; 20 ± 1; 24 ± 2; 22 ± 3), TXB2 (170 ± 15; 178 ± 11; 195 ± 11; 162 ± 22), U-BTG (0.18 ± 0.04; 0.17 ± 0.05; 0.26 ± 0.08) were not different from normal. Gp II; P-BTG (38 ± 5; 35 ± 4; 38 ± 4; 39 ± 5) TXB2 (525 ± 139; 358 ± 40; 356 ± 37; 330 ± 34) were different from Gp I on all days (p<0.01); U-BTG (0.38 ± 0.13; 0.14 ± 0.03; 0.14 ± 0.03) was not different from Gp I. Gp III; P-BTG (49 ± 12; 33 ± 3; 36 ± 5; 29 ± 3) and TXB2 (237 ± 32; 289 ± 39; 285 ± 38; 340 ± 57) were elevated compared to Gp I (p<0.01), but not to Gp II, except for TXB2 at presentation (p<0.05); U-BTG(0.49 ± 0.19; 0.40 ± 0.11; 0.16 ± 0.05) was not different from either Gp I or II. In Gp II, TXB2 levels were higher (p<0.01) in 26 patients with recurrent episodes of ischemic pain (636 ± 238; 439 ± 56; 419 ± 51; 406 ± 42) compared to 21 stable patients (413 ± 149; 243 ± 46; 264 ± 48; 216 ± 39). Plasma BTG was normal in 61% of patients with established CAD, and the correlation with clinical course was poor. There was a weak correlation between P-BTG and TXB2 (r= 0.20, p<0.01) and P and U-BTG (r= 0.31; p<0.01). These data indicate that platelet release and prostaglandin synthesis occur in a proportion of patients with coronary ischemic syndromes. TXB2 is more often increased than BTG and occurs in most patients with recurrent ischemia. However, neither test has sufficient sensitivity to be of clinical value.
The Effect Of Prostacyclin On The Participation Of Platelets In X-Activation And Thrombin Formation
Authors: Bevers E , Dieijen G V , Rosing J , Hornstra G , Zwaal R F A
Published: July 2018

Damaged vascular tissue triggers the intrinsic and extrinsic clotting system, resulting in the clotting of plasma. Platelets, especially after their activation, promote this clotting response, which is inhibited by prostacyclin (PGI2).The present experiments were devised to characterize this anticoagulant effect of PGI2.

Using specific chromogenic substrates, the intrinsic activation of factor X and the conversion of prothrombin into thrombin (IIa) was measured in reaction mixtures containing highly purified clotting factors. Phospholipids were added as vesicles, platelet lysates, or as whole platelets, either or not activated with a mixture of collagen and thrombin. Phospholipid vesicles and lysed platelets greatly promoted Xa and IIa formation, which was not affected byPGI2.The very low formation of Xa and IIa occurring in the presence of non-activated platelets was not inhibited byPGI2 either. Platelets activated with a mixture of collagen and thrombin stimulated Xa and IIa formation considerably. PGI2 inhibited this effect in a dose-dependent way.

These results demonstrate that prostacyclin does not interfere with Xa and IIa formation as such, but specifically inhibits the process by which a mixture of collagen and thrombin stimulates the participation of platelets in these reactions.

The inhibiting effect of PGI2 is only partial. This is most probably explained by the fact that prostacyclin can only inhibit but not block platelet activation by a mixture of collagen and thrombin.
The Effect Of Prostacyclin (PGI2) On The Procoagulant Activity Of Human Platelets
Authors: Brox J , Østerud B
Published: July 2018

Platelets from healthy donors were isolated by albumin- gradient centrifugation and gelfiltration. The platelets were exposed to thrombin, collagen, and ADP separately, and thrombin and collagen in combination. The concentrations used were the lowest that gave maximal aggregation. The following parameters were assayed: aggregation, platelet factor 3(PF 3), Factor V-Va(F.V-Va), total procoagulant activity (TPA, which measures the combined activity of PF 3 and F.V-Va), and serotonin release. The effect of various concentrations of PGI2 on these parameters was examined.

Thrombin was more potent than collagen, and collagen was more potent than ADP in stimulating the procoagulant activity and serotonin release (i.e. thrombin generated 33%, collagen 14%, ADP 3% TPA as compared to 100% for lysed platelets). Thrombin alone was equally strong as thrombin and collagen in combination in regard to TPA. The platelet aggregation was maximal in all these experiments.

PGI2 (1.4x10-8M) inhibited very efficiently aggregation, serotonin release, TPA, PF 3 and F.V-Va activity when the platelets were stimulated with thrombin, collagen or ADP. When platelets were exposed to thrombin and collagen simul- tanously, the inhibitory effect of PGI2 on TPA decreased. PGI2 concentration of 1.4xlO-7M in such platelet mixtures inhibited TPA by 30-40% whereas the same PGI2 dose inhibited TPA by 80-90% when thrombin and collagen were used separately. In these experiments platelet aggregation was less than 20%.

This study demonstrates that PGI2 strongly inhibits the availability of the platelet procoagulant activity, and PGI2 may therefore also slow down the generation of thrombin.
Reduced Production Of 6-Ket0-PGF1α By Umbilical Artery In Pre-Eclampsia
Authors: Carreras L O , Van Assche A , Defreyn G , Van Houtte E , Vermylen J
Published: July 2018

Decreased production of PGI2 activity by fetal and maternal vessels and low values of PGI2 activity in amniotic fluid have recently been observed in patients with severe pre-eclampsia. In this study we measured the production of 6-keto-PGF1α by the umbilical artery and placental veins in 5 cases of severe pre-eclampsia (diastolic blood pressure > 100 mmHg) and 5 normal pregnancies. No mother had ingested aspirin for at least a week before delivery. Vascular specimens were obtained immediately after expulsion of the placenta, immersed in tris buffer 0.05 M, pH 7.4 and cut into fine rings. The production of 6 keto-PGF1α , was studied within 2 hours. Vascular fragments were incubated on a gentle rocker platform at 22°C in 2.5 ml of the following media :
a) tris buffer ; b) tris buffer containing sodium arachidonate (AA) 5 pM. 100 pi subsamples were removed from the incubation dishes at 0, 4, 8, 12, 16 and 24 minutes. The 6-keto-PGF1α level in each of the subsamples was measured by radioimmunoassay. The mean values obtained, expressed as pg.mgcr-1± SEM.

A significant reduction in the production of 6-keto-PGF1α by umbilical artery incubated in tris buffer (p<0.01) and tris buffer containing sodium arachidonate 5 pM (p< 0.025) was observed in the cases of severe pre-eclampsia. In contrast, no significant decrease in the production of 6-keto-PGF1α by placental veins was obtained. Considering that fetal umbilical arteries generate much greater PGI2 activity than vessels from normal adults, a central role for this substance in the regulation of fetal circulation has been postulated. The marked reduction in the production of PGI2 could seriously affect fetal outcome in severe pre-eclampsia.
Prostaglandins And Uterine Bleeding
Authors: Goodfellow C F , Paton R C , Moncada S , Salmon J A , Davies J A , McNicol G P
Published: July 2018

There is little information about haemostatic function in the uterus in relation to menstrual bleeding. We measured platelet count and platelet retention in glass-bead columns, and plasma levels of FPA, TxB2 and 6-oxo-PGF1α in samples taken simultaneously from uterine vein (UV) and peripheral vein (PV) in 18 women (2 post-menopausal) undergoing hysterectomy. Platelet counts and FPA concentrations were similar in both sets of samples. Plasma levels of 6-oxo- PGF1α were significantly (p<0.01) higher in UV (1.34 ± 1.27 ng/ml, mean ± SD) than in PV (0.18 ± 0.39 ng/ml) as were levels of TxB2 (0.46 ± 0.54 and 0.1 ± 0.13 ng/ml). Platelet retention in UV blood was about half that observed in PV blood and the degree of platelet retention correlated inversely with plasma concentration of 6-oxo-PGF1α (r = -0.48, p<0.01). There was a highly significant (p<0.01) rank order between the time since menstruation and plasma 6-oxo-PGF1α concentration in UV blood. TxB2 levels were highest on day 4 and then fell suddenly with time since menstruation. The results indicate that prostaglandins may modulate uterine bleeding at menstruation. Release of PGI2 from uterine vessels could contribute to the lack of platelet deposition in the menstruating uterus and by this mechanism to the persistence of menstrual bleeding.
Inhibitory Effect Of Exogenous Arachidonic Acid Or Linoleic Acid On Rabbit Platelet Aggregation And Release Reaction
Authors: Cattaneo M , Kinlough-Rathbone R L , Mustard J F
Published: July 2018

In contrast to other release-inducing agents (e.g. thrombin) arachidonic acid (AA) releases only 40-50% of amine storage granule contents and although low concentrations induce aggregation, high concentrations do not. Several theories have been proposed to explain these observations: 1) AA or its products inactivates the cyclo-oxygenase; 2) the products of AA increase platelet cAMP; 3) lipoxygenase products are inhibitory; 4) unsaturated fatty acids (UFA) perturb the cell membrane. Using washed rabbit platelets we examined the effect of AA on platelet function. In these experiments aspirin-treated platelets (ASA 5.5 mM) were exposed to AA (230 μM) for 15 min. and then to PGEj (10 μM) for 30 min. The platelets were then resuspended. These platelets did not aggregate to ADP (9 μM) and their response to thrombin (0.02-0.05 U/ml) was impaired in contrast to control, ASA-treated platelets not exposed to AA. Non-ASA-treated platelets exposed to AA (230 μM), deaggre- gated with PGE1, and then resuspended also did not aggregate in response to ADP (9 μM) collagen, AA (230 μM) or thrombin (0.02-0.05 U/ml). When platelets pretreated with ASA and AA were mixed 1:1 with normal platelets and the mixture stimulated with AA (230 μM), the AA-treated platelets did not release their granule contents whereas the normal platelets did. These results do not support the hypothesis- that the inhibitory effect of AA on platelet aggregation and release is primarily due to inhibition of cyclo-oxygenase or an increase in cAMP caused by AA products. It seems unlikely that inhibition by AA can be due to products of the lipoxygenase pathway, because the effect persists when the platelets are washed and resuspended. Similar results were obtained by incubating platelets with linoleic acid (230 μM). This evidence is compatible with the hypothesis that UFA can inhibit platelet function by perturbing the cell membrane. This effect may be related to changes in receptor availability.
In Vitro And In Vivo Effects Of An Inhibitor Of Thromboxane Synthetase
Authors: Defreyn G , Carreras L O , Machin S J , Vermylen J , Verstraete M
Published: July 2018

UK-37,248, 4-(2-(lH-imidazol-l-yl)ethoxy)benzoic acid hydrochloride, completely inhibits platelet aggregation in plasma by low concentrations of arachidonic acid at 0.5 uM and thromboxane B2(TXB2) generation in washed platelets at 10 uM. In the latter test system the total amount of cyclooxygenase metabolites is unaltered, the decrease in TXB^ and hydroxyhepatadecatrienoic acid being compensated by an increase in prostaglandins E2 and F2α . Arachidonic acid challenged platelets pretreated with UK-37,248 do not accumulate cyclic AMP ; they however strongly stimulate the production of prostacyclin by aspirin pretreated cultured endothelial cells.

In a double blind placebo controlled study ingestion of 200 mg of the compound resulted in a complete inhibition of arachidonic acid induced platelet aggregation, whereas the threshold concentration for irreversible platelet aggregation with ADP was unaltered. Serum TXB2 levels were markedly decreased from the normal pre-values (200-700 pg/ml) to low (60-80 pg/ml). Stimultaneous plasma 6-keto prostaglandin F2α , levels from citrate blood increased from 46 ± 23 pg/ml (mean ± SD) to 409 ± 185 pg/ml).

It is concluded that a thromboxane synthetase inhibitor modifies cyclic endoperoxide metabolism in such a way that there is not only a decreased formation of pro-aggregatory thromboxane A2 but also an increased production of antiaggregatory prostacyclin. Thromboxane-synthetase inhibitors may be superior to aspirin as antithrombotic agents. The platelet function defect induced by UK-37,248 is only distinct from that in congenital thromboxane synthetase deficiency in that in the latter condition ADP aggregation always is reversible and arachidonic acid challenged platelets accumulate cyclic AMP (Defreyn et al, 1981). Probably UK-37,248, like imidazole itself, activates phosphodiesterase, thus abolishing part of its anti-aggregating effect.
Identification Of The AT III Synthesizing Hepatocytes By Immunofluorescent Technique
Authors: Watada M , Nakagawa M , Kitani T , Okajima Y , Maeda Y , Urano S , Ijichi H
Published: July 2018

Antithrombin III (AT III), heparin cofactor has been known to be one of the important protease inhibitors on the regulatory mechanism of coagulation and fibrinolysis, but the site of synthesis of plasma AT HI has not yet been conclusively established, although some observations suggested that the major site of AT III production may be the liver and AT III levels in plasma is known to be decreased on the hepatic disorders. This paper reports the deposition of the AT III specific fluorescence in the hepatocytes of rat liver by immunofluorescent examination. Plasma AT III was purified by heparin sepharose affinity chromatography and gel filtrations. Rabbit was immunized with this purified AT III and Freund’s complete adjuvant and rabbit anti rat AT III serum was obtained. This antiserum was comfirmed to be monospecific to rat AT HI by means of immunoelectrophoresis. Freshly isolated rat liver was prepared as specimen and fixed in acid alcohol and processed to dehydration and embedding into paraffin in conventional way. Indirect immunofluorescent technique with FITC labeled anti rabbit gamma-globulin were utilized for immunofluorescent staining and histological observation was performed under Nikon FL fluorescent microscope. The localization of fluorescence was observed in the hepatocytes which scattered in the acinus structures. The fluorescence in the cytoplasma of these hepatocytes was homogeneously stained. As far as the fluorescence is concerned, the synthesis of AT III may be performed not synchronizing among hepatocytes. This immunofluorescent study supports the reported data of hepatic perfusion.
Antithrombin III And Plasminogen Levels In Moderate And Severe Trauma
Authors: Dombrose F A , Seyfer A E , Callahan J
Published: July 2018

Functional and immunologic levels of antithrombin III (ATIII) and plasminogen were assessed in three groups of nine patients each: elective surgery, moderate and severe trauma. Patients otherwise healthy were classified as follows: I, elective extremity surgery, no blood transfusions, normal coagulation screening tests (14-50y); II, isolated, moderately severe trauma (e.g., replantation or revascularization of one or more digits), no transfusions (14-56y); and III, Isolated, severe trauma to extremities, with replantation and revascularization of a severed arm or leg, receiving 1-4 units of whole CPD blood (20-59y). Preoperative, intraoperative (2 hr into surgery) and 24 hr postoperative values were measured. Group III only had intraoperative sampling, with the exception of two patients who had preoperative values prior to any transfusions. A detailed statistical comparision was made (at the 95% confidence level) of the differences in ATIII activity, ATIII antigen level, the ratio of ATIII activity-to-antigen and plasminogen activity among categories within and between groups. The data suggest that elective surgery and moderate trauma had virtually the same effect on ATIII and plasminogen. Activity and antigen values decreased intraoperatively but on the average not enough to be out of the acceptable range. During the convalescent period most ATIII values returned to their preoperative level while most plasminogen values continued to decline somewhat (although still in the acceptable range). By contrast, both ATIII and plasminogen values in the severe trauma group were markedly depressed compared to preoperative levels In either group I or II. Four of the patients in this group suffered postoperative thrombosis and loss of an extremity even though the limb was initially viable. The transient lowering of both ATIII and available plasminogen is undoubtedly most critical when surgical stress is superimposed on existing trauma.
Aggregating Activity Of Structural Analogues Of Platelet Activating Factor (PAF): High Stereospecificity
Authors: Baumgartner H R , Hirth G , Michael C , Saroka H , Bannwarth W , Barner R
Published: July 2018

12 structural analogues of PAF, a 1-0-alkyl-2-0-acetyl-sn-glyceryl-3-phosphorylcholine, were synthesized in order (1) to study their structure-activity relationship and (2) to find competitive inhibitors of PAF.

The compounds were dissolved in ethanol and diluted with 0.1 % albumin in isotonic saline. They were added to stirred platelet-rich plasma (PRP) obtained from citrated blood. For each PRP the proaggregatory EC50 for ADP and 1-0-octadecyl-2-0-acetyl-sn-glyceryl-3-phosphorylcholine (Ro l4-8l6l) was determined using a Born MK3 or an elvi 840 aggregometer.

In human PRP Ro l4-8l6l had an EC50 of 43 ± 5 nM (mean ± SE, n = 6) and was 33 × more potent than ADP. Ro l4-8l6l was 160 x (n = 20) more potent than ADP and 1300 x (n = 6) more potent than its S-enantiomer in aggregating rabbit platelets (EC50 for Ro l4-8l6l = 3.8 ± 0.3 nM). Rabbit PRP was used to compare additional analogues. Introduction of a double bond (cis-9-octadecenyl) in the alkyl chain of racemic 1-0-alkyl-2-0-acetyl-glyceryl-3-phosphorylcholine caused a threefold increase of the aggregating activity. With the replacement of the acetyl group by H or CH3 in Ro l4-8l6l platelet aggregating activity virtually disappeared. Replacement of choline by ethanolamine caused considerable loss of activity. Permutation of the substituents attached to the glycerol moiety was also associated with partial loss of activity. Plasmalogen was inactive. None of the analogues inhibited Ro l4-8l6l induced aggregation.

Our findings demonstrate that the platelet aggregating effect of the PAF molecule is highly stereospecific: the natural configuration at C (2) of l-0-alkyl-2-0-acetyl-glyceryl-3-phosphorylcholine is of particular importance for activity.
In Vitro Effect Of Ticlopidine On Arachidonic Acid Metabolism In Platelet Phospholipids
Authors: Chap H , Simon M F , Douste-Blazy L
Published: July 2018

The effects of ticlopidine (Ti) on arachidonic acid (AA) metabolism in platelet phospholipids have been studied in vitro by following AA release from (14C) AA-prelabeled platelets or by measuring (14C)-AA incorporation into platelet phospholipids.

In the presence of prelabeled platelets, Ti induced a release of AA essentially from phosphatidylcholine (PC) only at concentrations (10-3 M) where the drug became lytic. Incubation of cells previously lysed by sonication led to a deacylation of PC, part of the released AA being reincorporated into phosphatidylethanolamine (PE) . Under these conditions, Ti effect on PC disappeared and only a slight inhibition of AA incorporation into PE was observed.

On the other hand, upon incubation of non-labeled platelets with (14C)-AA, Ti impaired the incorporation of AA into all platelet phospholipids, half maximum effect being observed under non-lytic conditions at 10-5-5.10-5 M.

It is postulated that Ti inhibits the acylation reactions responsible for AA entry into glycerophospholipids. This effect might promote the release of trace amounts of AA in intact cells, which could explain the accumulation of stable, anti-aggregating prostaglandin D2 (M. Lagarde et al. Prostagl. Med. 1979, 2, 433).
Estrogen Therapy Induced Antithrombin III Decrease And Deficiency In Patients With Prostatic Cancer
Authors: Büller H R , Boon T A , Dabhoiwala N F , Henny Ch P , ten Cate J W
Published: July 2018

Plasma AT III activity was studied in 12 patients with cancer of the prostate receiving estrogen therapy in different dosages. AT III was determined with an automated chromogenic method (normal value 0.80-1.40 u/ml). In six patients with maintenance diethylstil- bestrol (DES) therapy(2.5 mg/48 hrs) mean plasma AT III activity was normal (X 0.88 u/ml, range 0.74-1.05 u/ml). The dose of DES at 15 mg. daily resulted in a marked AT III activity decrease (mean 0.24 u/ml) in all six patients. In patients with low normal initial plasma AT III activity acquired AT III deficiency, with enhanced risk of thromboembolic complications, may develop. The observations suggest that plasma AT III activity should be monitored before and during estrogen therapy in prostatic cancer patients. In patients with acquired AT III deficiency ( <0.80 u/ml) prophylactic administration of low dose heparin subcutaneously should be considered.
Determination Of Antithrombin III In Extravasal Body Fluids By Radioimmunoassay
Authors: Bleyl H
Published: July 2018

Detection of trace amounts of AT III need highly sensitive methods like a radioimmunoassay. Purified human AT III was radiolabeled enzymatically with the GOD/POD method. After iodination, the 125 J-AT III was purified by affinity chromatography over heparin agarose. The antibody (rabbit immunoglobulin) was adsorbed to protein A-Sepharose and covalently linked by dimethyl suberimidate. Binding capacity of the radioiodi- nated AT III was >90%. The sensitivity of the immunoassay was 20 ng/ml AT III. The intercept point (50% displacement) was at 300 ng/ml AT III. Healthy volunteers with a renal excretion of < 50 mg/24 hours total protein had an AT III excretion of 30 - 50 ug/24 hours.

< 50 mg/24 hours total protein had an AT III excretion of 30 - 50 ug/24 hours.

Spinal fluid within the normal range of 150 - 450 mg/1 total protein contains 0.5 - 2.5 mg/1 AT III.

Patients undergoing continous ambulant peritoneal dialysis (CAPD) lost 2.5 - 12 g protein with up to 4×1.5 - 2 l dialysate. The loss of AT III was 20 - 100 ng/24 hours.
Complement Activation And C1q Binding Activity In Haemodialysis
Authors: Dodd N J , Vergani D , Turney J H , Bevis L , Weston M J
Published: July 2018

It has been proposed that hypoxia occurring during Haemodialysis (HD) is related to complement activation by the dialysis membrane. Consequent leucocyte aggregation may then lead to pulmonary micro-embolisation. We set out to assess complement activation during HD using a new, sensitive, assay for activation fragment C3d.

Twenty patients, established on long term HD had blood samples taken prior to, and after 60 and 120 minutes on dialysis. C3 and C4 were measured by conventional nephelometric assays,and C3d by a new nephelometric assay. We also measured C1q binding activity (C1qBA).

Hypoxia was maximal at 60 minutes (73.9% of pre-dialysis value). Pre-dialysis C3d levels were elevated,and rose significantly on HD at 60 and 120 minutes. C3 levels were persistently low-normal. C4 levels were normal and showed no change during HD. CIqBA increased during HD representing either liberation of pre-formed immune complexes(IC), or de-novo formation of IC, perhaps related to heparinisation.

C3d levels are a more sensitive indicator of complement activation than conventional complement component assays. (Previous studies have failed to demonstrate C3 or C4 depletion during HD). It is possible that HD induced complement activation may occur via the classical pathway, as a result of immune complex activity, or (more likely in view of normal C4 levels) via the alternative pathway, as a result of blood contact with the cuprophane membrane.

In addition to hypoxia, complement activation and C1qBA may lead to other recognised complications of regular HD including 1. Endothelial damage, 2. Hypersplenism and 3. Thrombocytopenia.
Prothrombin Activation By Factor B (Bb) Of The Alternative Pathway Of Complement
Authors: Fair D S , Sundsmo J S , Schwartz B S , Edgington T S , Müller-Eberhard H J
Published: July 2018

It has been reported that complement activation in serum by inulin, endotoxin, staphlococcal protein A, or heat aggregated γ-globulin results in consumption of prothrombin (Zimmerman and Müller-Eberhard, 1971, J. Exp. Med., 124;1601). The availability of purified complement proteins and coagulation factors prompted the study of complement factor(s) mediating prothrombin consumption. The ability of the classical and alternative pathway C3 convertase to cleave prothrombin was investigated by incubating 125I-labeled prothrombin (30min./37°C) with erythrocytes (E) bearing intermediate complexes of complement. Cleavage of prothrombin was analyzed by SDS-polyacrylamide gel electrophoresis and detected by autoradiography. 1) EC3b, Bb, NF; 2) EAC4oxy2,3b, Bb; and 3) Factor Xa were found to cleave prothrombin 43.2%; 20.2%; and 100% respectively. Prothrombin cleavage products generated by complement were similar to those produced by Factor Xa. The controls 1) EC3b; 2) EC3b, B; and 3) Edid not cleave prothrombin. Cells bearing the classical pathway C3-convertase EAC4oxy2 cleaved prothrombin at a slower rate (1.2%). Fluid phase C3-convertase formed with purified cobra venom factor (CVF), CVF, Bb, also cleaved prothrombin (28.7%). The precursor proteins of the enzyme, (CVF, B, or D) were incapable of cleaving prothrombin, whereas purified Bb, alone was active. Cleavage of prothrombin required Ca++ and Mg++. Cleavage by EC3b, Bb was inhibited by affinity purified goat anti-B immunoglobulin and by Trasylol. Both reagents were also found to inhibit Factor B hemolytic activity. These results suggest that activated Factor B (Bb), the central serine esterase of the alternative complement pathway, may cleave prothrombin directly without the intervention of Factor X. The observation of a complement pathway prothrombinase emphasized the integration of mediation pathways and suggests that previously unrecognized routes for initiating the coagulaion pathways may still exist.
A Simple And Rapid Method To Detect Platelet Associated IgG
Authors: Takahashi A , Ohara S , Imaoka S , Kambayashi J , Kosaki G
Published: July 2018

Anti-platelet antibody has been generally recognized as an important etiologic factor in idiopathic thrombocytopenic purpura (ITP) and the detection of platelet associated IgG (PAIgG) is essential to establish the diagnosis and also useful to predict the clinical course. Several methods to detect PAIgG have been proposed, most of which, however, require complicated manipulation and are not practical for clinical use. This paper describes a simple and rapid method to detect PAIgG.

This method was devised, utilizing the ability of staphylococcal protein A (SpA) to bind specifically to human IgG. An adhesive percentage of sample platelets to sheep red blood cells coated with SpA was calculated by enumeration of sample platelet before and after treatment. It takes only several hours to complete the assay from the drawing of blood. In a total of 37 cases including 15 cases of ITP, PAIgG was studied, using this method and Fab, anti-Fab assay of Luiken. Binding Index (an adhesive percentage) was 9.3 ± 3.8% in mormal subjects and 26.5 ± 10% in ITP patients, which differnce was statistically significant (p<0.01). Binding Index was within normal range in patients with non-immune thrombocytopenia such as liver cirrhosis or splenomegaly. A good correlation was observed between Binding Index and platelet count in ITP (r= -0.7299, p<0.01) and also between Binding Index and values of PAIgG by Fab, anti-Fab assay (r=0.8309, p<0.01).

These results suggest that this new method is reliable to detect PAIgG in addition to its simplicity and rapidity and practical in clinical use.
A Rapid And Sensitive Method For The Quantitation Of Platelet Autoantibodies
Authors: Jacks F , Bradlow B A
Published: July 2018

Autoantibodies were solubilized from washed platelets by three successive freeze thaw cycles followed by sonication for 15 seconds at maximum intensity (MSE Mk 2). The disrupted platelets were centrifuged at 30,000g for 20 minutes and the supernatant was heated at 56°C for 30 minutes followed by centrifugation at 3,000g for 5 minutes. An aliquot of the supernatant was diluted with 0,01M phosphate buffered saline pH 7.4 to give a final concentration equivalent to about 5 × 106 platelets/ml. Fifty microlitres of the diluted extract was adsorbed onto the wells of a microtitre plate by incubation at room temperature for two hours. The wells were then washed three times with 0,01M phosphate buffered saline pH 7.4 containing 0,05% Tween (Tween-PBS) and the adsorbed antiplatelet IgG reacted with 50μl of an iodine-125 labelled affinity isolated goat antihuman (heavy chain specific) IgG, specific activity ≃ 50μCi/ μg (California Antibodies) for one hour at room temperature followed by three washes with Tween-PBS. A standard curve was constructed and run in parallel, in the dose range 0,l-100ng using purified human IgG (Miles Laboratories). The separated wells were counted in an automatic gamma spectrometer (Packard 3003) and the results calculated on a desk-top computer (Hewlet-Packard 9800) using a Rodbart weighted Logit-Log transformation (f = 0,996). A relatively poor arithmetic correlation was found between whole blood platelet counts and measured platelet bound IgG (r = -0,42), whereas a better correlation was obtained using a log-log plot (r = - 0,84).

The results in ng/106 platelets were - Known ATP 133,2 ± 154, other autoimmune diseases with thrombocytopenia 212,0 ± 202, non-immune thrombocytopenia 18,6 ± 4,8, normal healthy controls 11,4 ± 3.7
Neonatal Polycythemia: A Disorder Associated With Hyperviscosity, Normal Coagulation Findings And Thrombocytopenia
Authors: Katz J , Rodriguez E , Madani C , Hicks D , Branson H E
Published: July 2018

Thirty-two newborns with elevated capillary hematocrits >65% were studied. Twenty-two newborns required plasmaexchange transfusion. All had central (venous) hematocrits >65% and had symptoms referrable to complications associated with this syndrome. Of the 22, 15 were appropriate-for-gestational age, 5 were small-for-gestational age, and 2 were large-for-gestational age. Viscosity measurements in the 10 newborns who did not require plasma-exchanges showed increased viscosity in 2 in the slow shear rates associated with bloodflow in the smaller vessels. Coagulation data before and after plasma exchange did not show a hypercoagulable state: PT-14.2±0.7 and 12.9±1.2 secs, PTT 49.9±3.6 and 42.2±3.2 secs, factor VII 73±5 and 78±5%, factor VIII 103±10 and 94±10%, AT III levels were low 14±1.2 and 17±1.3 mg/dl, fibrin degradation products were <10μg/ml, fibrin monomer was not detected, plasminogen levels were 5±0.8 and 7±0.9mg/dl, fibrinogen levels were 203±9.8 and 200±11.8 mg%. Vitamin K dependent factors were reduced factor V 44±6 and 49±11%, factor VII 77±5 and 86±5%, factor IX 28±2 and 42±3%, factor X 35±4 and 62±6%, factor XI 55±5 and 84±9%, factor XII 47±5 and 63±5%. Statistical significant differences were found only with factors IX, X, XI and XII. Thrombocytopenia was present in 6 patients (20% incidence) and post plasma exchange the platelet counts rose significantly and in 2 patients within 3 days reached normal levels. No statistical difference in the platelet counts were noted before and after the plasma-exchange and were similar to the levels determined in 10 newborn controls. Neonatal polycythemia with thrombocytopenia may indicate a more severe disorder, with hematocrits in the 6 patients >70%. It is suggested that the mechanism of the thrombocytopenia may be aggregates of platelets that deaggregate following plasmaexchange. The complications associated with neonatal polycythemia appear related to hyperviscosity, erythrocyte and platelet “sludging” in the smaller vessels.
Selectivity Increase Of Chromogenic Assay Of Factor Xa By Use Of Highly Selective Synthetic Thrombin Inhibitor Having Extremely Potent Stereostructure (No. 805)
Authors: Okamoto S , Ikezawa K , Nagano S , Matsuoka A , Hijikata A , Tamao Y
Published: July 2018

Chromogenic peptide substrates S-2222 designed for F. Xa assay is also amidolyzed relatively slowly by thrombin; in the usual F. Xa assay system however a considerable amount of thrombin is inevitably formed. In the present study, therefore, No. 805, highly selective synthetic thrombin inhibitor, (4 methyl- (methyl-tetrahydro-quino- linesulfonyl-arginyl ][ 2-piperidinecarboxylic acid), having extremely potent stereostructure (2R, 4R) at 4-methyl-2- piperidinecarboxylic acid portion, which was reported by S. Okamoto et al. in 1980, was used to improve selectivity of S-2222 for F. Xa assay. F. Xa in human plasma activated by either Russel’s viper venom or thromboplastin was estimated according to the methods by Aurell et al. . Ki of No. 805 for thrombin was found 0.019 juM and presence of No. 805 of 5 mM inhibited thrombin at least by 99.5%. However, No. 805 of this concentration showed no inhibition for purified F. Xa. Results obtained from chromogenic assay for F. Xa by S-2222 with or without No. 805 of 5 mM indicated that 10̴20% of amidolysis was derived from the action of thrombin formed in the assay system. Concerning high Km value of S-2222 to thrombin, molecular concentration of thrombin formed in the assay system was calculated as 3̴6 times of the F. Xa molecular concentration. Use of highly selective and potent synthetic thrombin inhibitor seems to be promissing to improve the selectivity of some other chromogenic assay also.
Interaction Of Factor XI With Activating And Non-Activating Surfaces
Authors: Mannhalter C , Schiffman S
Published: July 2018

Recent experiments have shown that purified human factor XI and trypsin activated factor XI adsorb not only to glass but also to several plastic materials. For both kinds of material the binding site(s) has (have) been localized in the heavy chain region of the molecule. Albumin (lo mg/ml) markedly reduces adsorption to plastics but not to glass, which could indicate different binding mechanisms to glass and plastics.

To help elucidate the nature of binding of factors XI and XIa to the different surfaces, three types of eluting agents have been evaluated for their ability to desorb bound factors XI and XIa -- high salt (2,4 M NaCl), a non-ionic detergent (Triton X-loo), and an anionic detergent (SDS).

High salt is only effective in eluting factors XI and XIa from glass and siliconized glass, which suggests ionic binding. Triton X-loo, which can split hydrophobic bonds, desorbs factor XI from plastics (except polycarbonate) and only poorly from glass. Elution with SDS, which has both a highly charged group and a hydrophobic hydrocarbon chain, is successful on glass and plastics.

We therefore conclude that the binding of factor XI to glass is the combined effect of ionic and hydrophobic binding, whereas the adsorption of factor XI to plastics is primarily hydrophobic.

Activation of factor XI, certainly a major change in the protein structure, appears to increase hydrophobic bonding to glass, since activated factor XI is almost completely eluted from glass by Triton X-loo.
Kinetics Of Adsorption Of Proteins From Human Plasma Onto Foreign Surfaces
Authors: Brash J L , Uniyal S
Published: July 2018

It is believed that adsorption of proteins is the first occurrence after blood/foreign surface contact. The composition of the protein layer, how it depends on surface properties, and how it changes with time are essentially unknown. The objective of this work was to develop data relevant to these questions. To this end, the quantities of �albumin, fibrinogen and IgG adsorbed on seven surfaces from human plasma as a function of time were measured. Human plasma (ACD anticoagulant) was diluted 1:4 with tris buffer. Purified proteins were labelled with iodine isotopes using the IC1 method and added to the plasma as tracers. Materials studied include several segmented polyether-urethanes, both hydrophilic and hydrophobic, glass, siliconized glass (SG), polystyrene (PS) and polyethylene (PE).

The results may be summarized as follows: Fibrinogen: Within the 2 min to 3 h range of contact times, fibrinogen was not detected on any of the hydrophilic surfaces. On PE and SG the quantity adsorbed passed through a maximum between zero time and 2 min, then declined to near zero. Only on PS was adsorption substantial (0.4 μg cm-2) and constant with time, similar to that from a solution of fibrinogen. Albumin: Albumin was also not detected on the hydrophilic materials. In general its surface concentration when it was adsorbed (hydrophobic surfaces) was similar to that observed for solutions of albumin. IgG: IgG was detected on all surfaces. The surface concentrations were low (about 0.1 μg cm-2) compared to solution values but were generally constant with time.

The following conclusions are drawn: (1) The plasma itself modifies adsorption. Therefore solution adsorption data cannot be used to predict plasma adsorption. (2) Contrary to popular belief, fibrinogen is absent or transient on most surfaces. (3) IgG appears to be ubiquitous as a component of protein layers adsorbed from Plasma.