bluuebunny/crossref_metadata_2025 huggingface dataset:
PROTEIN C IS ACTIVATED AND GIVES 110,000 MW COMPLEXES AND PROTEIN S IS CLEAVED AND DECREASED _IN VIVO IN PATIENTS WITH INTRAVASCULAR COAGULATION (DIC)
Authors: Heeb M J , Mosher D F , Griffin J H
Published: August 2018

Immunoblotting studies using denaturing and nondenaturing polyacrylamide gel electrophoresis conditions were performed on 100 plasmas from 88 patients with suspected DIC, in order to determine whether the anticoagulant regulatory proteins C and S (PC and PS) cure altered in vivo during DIC. 70 of these plasmas from 65 patients contained 5-35% of PC antigen in the form of activated protein C (APC) complexed with inhibitor(s).24 normal plasmas showed no detectable APC-inhibitor complexes.The complexes in DIC plasmas had a MW of 110 K on SDS-PAGE, as did complexes formed when APC was incubated with plasma immunodepleted of PC, or when PC in normal plasma was activated with Protac C. On nondenaturing gels, the complex present in 69 of 70 patient plasmas had the same mobility as one of two major bands of complexed APC observed in Protac C-activated normal plasma. One patient plasma contained two forms of PC antigen complex. This patient had suffered a perforated uterus during an abortion. After Protac C activation of the patient plasmas, two APC complexed bands were seen. The 16 patients with >15% complexed PC antigen included 3 with severe infection, 5 with solid tumors, 3 with leukemias, 2 with vascular disease and 3 with other diagnoses. These patients had a higher mortality (69%) than the group as a whole and higher levels of fibrin degradation products. 13 of these 16 plasmas and 56 of the entire group of 100 contained a higher than normal proportion of PS in a cleaved form with an apparent molecular weight lower than intact PS on reduced SDS-PAGE. Mean levels of PS antigen determined by electroimmunoassay for 95 of the plasmas were as follows: entire group, 86% (92%); patients with infection (n=34), 76% (81%); patients with malignancy (n=37), 102% (105%); all others (n=24), 70% (74%) wherfe numbers in parentheses exclude patients with liver disease and 100% normal pooled plasma. These studies suggest that PS is cleaved and decreased in vivo and that PC is activated in vivo and complexed with a 50K MW inhibitor(s) during DIC.
COMPARISON OF TO BIOLOGICAL EFFECT, PHARMACOKINETICS AND REPRODUCIBILITY OF INTRANASAL AND INTRAVENOUS ADMINISTRATION OF DDAVP
Authors: Lethagen S , Harris A S , Nilsson I M
Published: August 2018

DDAVP has previously mostly been given by the parenteral route but the development of intranasal delivery has made it possible for the patients to treat themselves at home without delay. The effect of intranasal administration of DDAVP as drops using a rhinyle catheter or a pipette has been unpredictable. The development of a spray has improved the intranasal administration of DDAVP with well controlled doses and better absorption.

In this study we conpared intranasal administration of 300 pg DDAVP by spray, with intravenous administration of 0.2, 0.3 and 0.4 pg DDAVP/kg in 10 healthy volunteers. We measured the effect on VIII:C, VIII:Ag and vW:Ag and also followed plasma levels of DDAVP before and 10' , 30' , 45' , 60' , 90' , 2 h, 4 h, 6 h and 8 h after administration.

We also studied the reproducibility of the spray effect. 10 healthy volunteers were tested before and 1 h after the administration of 300 pg DDAVP intranasally by spray on 5 different occasions with an interval of at least 1 week between the tests.

The highest response was obtained after 0.3 μg/kg i.v. The response to 0.4 μg/kg i.v. was less than 0.3 μg/kg indicating that maximum stimulation was reached with 0.3 μg/kg. The effect of spray approximates the 0.2 μg/kg response.

The reproducibility of the effect of the spray dose on VIII:C was 25% (coefficient of variation) and 33% for the intra-individual and inter-individual variation respectively. This corrpares favourably with the inter-individual variation after intravenous administration of 0.2 pg/kg (30%), 0.3 μg/kg (22%) and 0.4 μg/kg (35%). It was of interest to note that the intra- and inter-individual variation of pre-treatment basal levels of VIII:C was 14% and 17% respectively.

Intranasal DDAVP (300 μg) is as effective as 0.2 μg/kg intravenously and provides an accurate, reproducible and convenient alternative to parenteral administration.
DOES DESMOPRESSIN ACETATE REDUCE BLOOD LOSS AFTER CARDIOPULMONARY BYPASS SURGERY?
Authors: Rocha E , Llorens R , Paramo J A , Arcas R , Cuesta B , Martin Trenor A
Published: August 2018

It has been suggested that desmopressin acetate (DDAVP) administration reduces blood loss after cardiac surgery. We have investigated the effect of DDAVP administration in a doubleblind, randomized, prospective trial including 60 patients undergoing cardiopulmonary bypass surgery. Thirty patients received 0.3 ug/kg DDAVP and 30 patients a placebo. The infusion was administered in a 50 ml saline solution over 15 min when cardiopulmonary bypass had been concluded. Blood samples were taken before surgery, immediately before and 90 min after DDAVP or placebo administration, and 24 hours postoperatively. The following parameters were measured in each sample: hematocrit, hemoglobin, platelet count, VIII:C and factor VIII:vWF. Bleeding time was also measured before operation and 90 min after treatment administration. Blood loss and transfusion requirements were evaluated from the beginning of treatment until 72 hours after surgery. Results showed no significant differences neither in total blood loss (833 ± 363 ml in the DDAVP group vs. 907 ± 646 in the placebo group) nor in blood transfusion (1633 ± 676 ml in the DDAVP group vs. 1643 ± 720 in the placebo group). The prolongation of bleeding time and the decrease of factor VIII:vWF, 90 min after treatment, were significantly lower (p < 0.05) in the DDAVP group as compared with the placebo group. We conclude that DDAVP administration does not reduce blood loss in patients undergoing cardiopulmonary bypass surgery, which would suggest a more complex mechanism to explain the increased bleeding in these patients.
IMMUNOASSAY FOR FACTOR VIII-HEAVY CHAIN. AN INDICATOR FOR IMMUNE COMPLEXES DURING HIGH DOSE FVIII INHIBITOR TREATMENT
Authors: Nordfang O , Ezban M , Knudsen J B
Published: August 2018

Specificity studies have shown that most hemophilia A inhibitor antibodies are directed towards the light chain of coagulation factor VIII (FVIII). Thus, conventional immunoassays for FVIII-antigen (FVIII:Ag) presumably have reactivity for FVIII-Light Chain (FVIII-LC) . Our sandwich FVIII :Ag assay has been shewn to be specific for only FVIII-LC. We have now developed a specific immunoassay for FVIII-Heavy Chain (FVIII-HC) . This has made it possible to investigate the FVIII-HC content in hemophilia A plasma, and to study the expression of FVIII-HC in culture medium frcm transfected cell lines.

By adding purified FVIII-LC and FVIII-HC in coagulation inhibition assay, plasma frcm one of seven hemophilia A inhibitor patients was found to be reactive with both FVIII-LC and FVIII-HC. IgG frcm this plasma was used for a FVIII-HC specific inhibition radioimmunoassay. The polyspecific antibodies were coated to microplates with removable wells. The coated wells were incubated with test sample and with purified 125I-FVIII-HC. When normal human plasma pool contains 1 U/ml of FVIII-HC, the sensitivity of the assay was 0.004 U/ml.

For normal plasma and plasma frcm non inhibitor hemophilia A patients, FVIII-HC measurements correlated with FVIII:C and FVIII-LC measurements. However, after FVIII injection hemophilia A inhibitor patients in high dose FVIII treatment showed a much higher FVIII-HC content (1-5 U/ml) than FVIII-LC and FVIII:C (< 0.05 U/ml). These patients have previously been shown to have antibodies towards FVIII-LC. Therefore the antigen measurements indicate that inhibitor patients in high dose FVIII treatment have FVIII/anti-FVIII-LC immune complexes. These circulating immune complexes may be the mediator of an antibody dependent immune tolerance, during the high dose FVIII treatment.
INTERACTION OF THROMBIN AND FACTOR Xa WITH BOVINE VASCULAR ENDOTHELIAL CELLS, SMOOTH MUSCLE CELLS AND RAT HEPATOMA CELLS
Authors: Dryjski Maciej , Kuo Be-Sheng , Bjornsson Thorir D
Published: August 2018

The inhibition of thrombin as well as of factor Xa has been thought to occur primarily in plasma through the neutralizing action of the serine protease inhibitor antithrombin III (AT-III). However, inhibition of thrombin and Xa by this mechanism may not be sufficient for effective elimination of these clotting factors in states of increased coagulation activity. The potential role of the vascular endothelium in the inhibition of clotting factor activities has therefore received attention in recent years. The aim of the present investigation was to characterize the binding and inhibition of thrombin and factor Xa to the vascular endothelial cell (EC), smooth muscle cell (SMC) and rat hepatoma cell (RHC) in vitro, as well as to evaluate the effects of plasma constituents upon the inhibition of these factors. Purified bovine thrombin and factor Xa were used. The enzymatic activities of both factors were assayed using chromogenic substrates. The cells were exposed for 5 U/ml thrombin or 0.5 U/ml factor Xa. After 10 minutes incubation, the initial thrombin activity in the solution had decreased by about 20% in case of EC and SMC and about 11% when incubated with RHC. Thrombin activity recovered from the cell surface amounted to 0.02 U/cm2. When the cells with the surface bound enzyme were incubated with defibrinogenated plasma or AT-III for 30 seconds, only about 10% and 25-40%, respectively, of initial activity could be found. In similar experiments with factor Xa, after 10 minutes incubation, the initial activity in the solution had decreased by 10%. Factor Xa activity recovered from the cell surface was 0.001 U/cm2. After 30 seconds exposure to AT-III, no cell surface related factor Xa activity was recovered, whereas 10% of the cell surface activity was recovered after incubation with defibrinogenated plasma. It is concluded that thrombin and factor Xa are taken up and inhibited by EC, SMC and RHC cell surfaces in similar ratios suggesting that cell surface-mediated inactivation of activated clotting factors is not restricted to vascular wall cells. The inactivation of factor Xa was dependent on AT-III, however, the inactivation of thrombin was further promoted by an additional unidentified plasma constituent
VON WILLEBRAND FACTOR AS A REGULATOR OF ENDOTHELIAL CELL-DEPENDENT FACTOR Xa FORMATION
Authors: Koedam Joost A , Sixma Jan J , Bouma Bonno N , Stern David M , Nawroth Peter P
Published: August 2018

Factor Xa (FXa) formation on the endothelial cell surface involves a membrane protein which promotes assembly of the Factor IXa-VIII-X complex. Since Factor VIII (FVIII) can also interact with von Willebrand factor (VWF), which is both present in the plasma and expressed by endothelium, we examined the effect of VWF on FXa formation. When monolayers of conditioned endothelium were incubated with FIXa (2.8 nM), FVIII (0.1 unit/ml), and FX (65 nM), the rate of FXa formation could be decreased in a dose-dependent manner by addition of VWF. At 10 min of incubation, a VWF concentration of 5 µg/ml caused a 93% inhibition of FXa formation. Addition of a polyclonal antibody (F(ab')2) directed against VWF which blocks formation of the FVIII-VwF complex, increased endothelial cell-dependent Factor IXa-VIII-mediated activation of FX by 2− to 3-fold in the absence of exogenous VWF, indicating a role for endogenous VWF. Since no VWF was detectable using a sensitive radioimmunoassay in reaction mixture supernatants, endothelial cell-associated VWF was considered as a potential binding site for FVIII, thereby removing it from the reaction mixture. In addition, we found no effect of either exogenous VWF nor anti VWF-antibodies when FVIII was activated with thrombin before starting the incubation.Radioligand binding studies were carried out with 125I-FVIII and demonstrated binding to a limited number of sites on intact endothelial cell monolayers which could be partially blocked by anti-VWF F(ab')2. These results suggest that VWF may regulate FXa formation on the endothelial cell surface.
EXTRINSIC ACTIVATION OF HUMAN COAGULATION FACTORS IX AND X ON THE ENDOTHELIAL SURFACE
Authors: Bom V J J , van Hinsberqh V W M , Reinalda-Poot J H , Mohanlal R W , Bertina R M
Published: August 2018

In previous studies using purified human tissue factor (TF) apoprotein reconstituted with PS/PC membranes, no dramatic differences between the kinetic parameters for extrinsic activation of factor X (FX)and factor IX (FIX)could be observed. In vivo however, TF is an integral part of a complex biological membrane. Therefore we studied the kinetics of extrinsic FX and FIX activation on endothelial cells.

TF expression was stimulated by incubation of cultured human endothelial cells with endotoxin (4 hr). The cells were washed and incubated with FVII(a), FX and/or FIX n the presence of CaCl2. FXa and FIXa formation were measured with a sensitive spectrophotometric assay (S2337) and an immunoradiometric assay (IRMA-IXa), respectively. Unstimulated cells did not induce significant activation of FX or IX. Stimulation of the cells and recombination of exposed TF withFVIIa resulted in rapidactivation of both substrates.Rates of FXa formation reached a maximum after a lag of about 1 min. Upon prolonged incubation the rate of FXa formation progressively decreased, probably by inactivation of FVIIa by product Xa. At the other hand, FIXa formation was linear in time for at least 30 min. When added together, FIX was found to be a weak, competitive inhibitor of FX activation, while FIX activation was severely inhibited by FX. Both FX and FIX activation were dependent on the presence of TF (sensitive to µ-TF antibodies) and FVIIa. The calculated Km of FIX (∼0.09µM) and of FX (∼0.07µM) for extrinsic activation at the endothelial surface were both higher than those observed in a cell free system using purified TF apoprotein and the ratio of Km-FIX/Km-FX was about 6-fold increased. The ratios of Vmax-FIX activation/Vmax-FX activation for both systems were similar (−0.3). These data indicate that the microenvironment of TF in the biological membrane does not introduce important alterations in the kinetic parameters of TF-FVIIa dependent activation reactions in favour of extrinsic FIX activation.
FIBRINOGEN BINDING TO ENDOTHELIAL CELLS AND INTERFERING PEPTIDES
Authors: Charon M H , Tranqui L , Andrieux A , Hudry-Clergeon G , Marguerie G
Published: August 2018

Fibrinogen interacts with platelets and endothelial cells via specific binding sites. While the platelet fibrinogen receptor has been identified and was found to be associated with GPIIb-IIIa, the binding site on endothelial cells has not been characterized yet. The platelet GPIIb-IIIa belongs to the newly identified cytoadhesin family which includes immunologicaly related receptors interacting with RGD containing proteins. A cytoadhesin has recently been described on endothelial cells and the possibility that fibrinogen might interact with this glycoprotein was examined. Peptides corresponding to γ and α chains sequences were synthesized and their capacity to inhibit fibrinogen binding to endothelial cells and platelets were compared. Analogues of the γ chain from His 400 to Val 411 produced an inhibition similar to that observed for fibrinogen platelet interaction, suggesting that the structure function relationship of γ peptides is identical in both systems. In contrast synthetic analogues corresponding to the a chain and including RGD yielded slightly different results. While RGD alone was inactive on platelet, this tripeptide was active on endothelial cells. RGDS and RGDF corresponding to α 572-575 and α 95-98 partially or fully inhibited fibrinogen binding to endothelial cells but the structure activity relationship was different when compared to that observed for platelets. Addition of the N and C sequences adjacent to RGDS reduced the activity of this peptide whereas the activity of RGDF analogues was not modified by addition of N and C-sequences. In contrast platelet fibrinogen binding decreased with these RGDF analogues. These results suggest that fibrinogen endothelial cell binding is mediated by an RGD adhesion receptor with subtle differences in the recognition selectivity of the ligand, which is controled by the surrounding sequence.
FIBRINOGEN SYNTHESIS BUT DEFECTIVE STORAGE IN THE PLATELETS OF A PATIENT WITH GLANZMANN’S THROMBASTHENIA
Authors: Belloc F , Hourdille P , Boisseau M , Nurden A T
Published: August 2018

Patient A.M. has a lifelong history of bleeding episodes and platelet function defects typical of type I Glanzmann's thrombasthenia. Analysis of platelet membrane glycoproteins (GP) by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) or by studying the binding of monoclonal antibodies to intact platelets, confirmed the presence of only trace amounts of GP lib and GP Ilia. SDS-PAGE also revealed a severe deficiency of the alpha-granule pool of platelet fibrinogen although an immunoblot performed using a monospecific rabbit anti-fibrinogen antibody showed the presence of residual amounts of fibrinogen. This was estimated to be approximately 10 % of the normal range by an ELISA procedure. Incubation of washed platelets from A.M. with (35S) methionine for 3 h at 37°C resulted in the incorporation of radioactivity into multiple protein bands as revealed by SDS-PAGE followed by fluorography. Immunoprecipitation experiments with affinity purified anti-fibrinogen IgG bound to ultrogel confirmed the synthesis of fibrinogen. The amounts of radioactivity obtained were similar to those immunoprecipitated from control platelet extracts under the same conditions. However, unlike for control platelets, the neosynthesized fibrinogen in A.M. platelets was no longer detected after a 18 h cold chase. In contrast, neosynthesized thrombospondin of the patient was normally preserved during the same chase period. When the fate of the neosynthesized fibrinogen in A.M. platelets was studied it was found to be lost at a faster rate than in normal platelets. Immunoblotting experiments confirmed that cytoplasmic proteases may degrade fibrinogen when exposed to the protein. Our results suggest that the fibrinogen deficiency in the platelets of patients with type I thrombasthenia may be related to a storage abnormality in megakaryocytes or platelets and not from a synthesis defect.
PARTIAL PLATELET FUNCTION DEFECT IN A VARIANT OF GLANZMANN'S THROMBASTHENIA WITH INTERMEDIATE LEVELS OF GP IIb/IIIa
Authors: Hardisty R M , Pannocchia A , Mahmood N , Nokes T J C , Pidard D , Bouillot C , Legrand C , Nurden A T
Published: August 2018

A 17-year-old Italian boy has had a lifelong bleeding tendency, with frequent epistaxes and gum bleeding. The bleeding time is prolonged and the platelet count low normal. Electron microscopy showed a wide diversity of platelet size with many giant forms. In citrated PRP, ADP and other agonists induce slow and incomplete aggregation. The response of washed platelets varied with the agonist but ranged from subnormal to almost normal. Fibrinogen binding to washed platelets occurred slowly in response to ADP but eventually approached normal levels. No significant abnormality was observed of 5HT uptake, adenine nucleotide content, platelet factor-3 availability, β-thromboglobulin content or release, or malonyldialdehyde production. Clot retraction was normal. SDS-PAGE showed reduced amounts of GPIIb and GPU Ia. Crossed immunoelectrophoresis of Triton X-100 extracts of washed platelets showed the presence of GPIIb/IIIa complexes at 25-50% of normal levels. SDS-PAGE combined with an immunoblot procedure confirmed unchanged mobilities of GPIIb and GPIIIa and a normal proportion of GPIIb to GPIIIa. However, binding studies with radiolabelled monoclonal antibodies showed that intact washed platelets expressed only 12-20% of the normal binding sites for M148, AP-2 and Tab. These antibodies recognize different epitopes on GPIIb/lIIa complexes. Similar levels of these glycoproteins were detected by autoradiography after SDS-PAGE of radio-iodinated patient's platelets. GP lb was normally present. A possible defect in the exposure of fibrinogen binding sites might contribute to the altered platelet function. Meanwhile, the patient appears to be a unique variant of Glanzmann's thrombasthenia with GP IIb/IIIa complexes at the borderline of those able to support platelet aggregation.
IMMUNE THROMBOCYTOPENIA (ITP) : PLATELET TARGET ANTIGENS OF THE ANTIBODIES IN DIFFERENT CLINICAL SETTINGS
Authors: Beardsley D S
Published: August 2018

Autoantibodies against platelet antigens are important in the pathogenesis of ITP. We have studied the antigenic targets of these autoantibodies by immunoblotting using electrophoretically separated proteins from normal, Glanzmann's Thrombasthenic, and Bernard-Soulier Syndrome platelets immobilized on nitrocellulose paper. Incubation of these proteins with ITP patient serum, immunoglobulin, or F(ab*>2, followed by labeled antiglobulin, allowed identification of the antigenic target glycoproteins (GPfs). Patients with ITP of several categories were studied: A) Chronic ITP (> 1 yr duration) including a subset of patients who hadmild thrombocytopenia but significant hemorrhagic manifestations. B) Acute ITP (<6 mo) following varicella infection

Acute onset ITP unresponsive to any of the usual therapeutic modalities. A majority of patients in group A (32/48) had antibodies directed against a protein with a MW of lOOkD. In some cases, the target antigen was localized to GPIIIa specifically by absence of reactivity with Type I Glanzmann's thrombasthenic platelets known to be totally deficient in GPIIb and Ilia. Two individuals in the subset with unexpectedly severe hemorrhagic symptoms also had anti-GPIIIa antibodies, but the antigenic target was different since only these two antibodies could be shown to interfere with binding of 125j_fibrinogen to normal platelets

In contrast to the individuals with anti-GPIIIa antibodies, all group B patients (7/7) with acute post-varicella ITP had antibodies directed against an 85kD thrombin sensitive protein, and two of the three group C individuals studied showed antibodies directed against GPIb. These studies indicate that a number of different platelet antigens can be the targets of platelet autoantibodies in ITP. Results suggest that antibodies against a particular antigen may correlate with a similar clinical setting in different patients. Further work will determine whether antigen identification at the time of diagnosis can be used to predict the clinical course for an individual patient
MECHANISMS OF IMMUNOLOGIC THROMBOCYTOPENIA IN INDIVIDUALS AT RISK FOR AIDS
Authors: Karpatkin S
Published: August 2018

HIV-seropositive homosexuals, narcotic addicts and hemophiliacs develop a new syndrome of immunologic thrombocytopenic purpura (ITP) which is clinically indistinguishable from classic autoimmune thrombocytopenic purpura (ATP) with respect to increased megakaryocytes in the bone marrow, peripheral destruction of antibody-coated platelets, negative serology for SLE, response to treatment with prednisone and/or splenectomy. However, their platelet immunologic profiles are different.

Homosexuals appear to have an immune complex-mediated mechanism: markedly elevated platelet-bound IgG and C3C4 (3.8 and 4.2-fold greater than classic ATP, respectively), elevated circulating immune complexes (3-fold greater than classic ATP), anti-F(ab')2 antibodies and absence of 7S anti-platelet IgG. There is no inverse correlation between platelet count and platelet-bound IgG or platelet-elutable anti-platelet antibody as in classic ATP.

Hemophiliacs appear to have an autoimmune 7S IgG-mediated mechanism similar to classic ATP: inverse relationship betweem platelet count and platelet-bound IgG, r = 0.84, p less than 0.001, 26 df, anti-platelet reactive 7S IgG which reacts by its F(ab')2 domain, (reactive at 60-130 ug/ml compared to control IgG), platelet-elutable anti-platelet antibody. However, these patients also have elevated circulating immune complexes (2.4-fold classic ATP level) and markedly elevated platelet-bound IgG and C3C4 (3.4 and 1.2-fold classic ATP level, respectively). Anti-HIV antibody correlated with circulating immune complexes, r = 0.833, p less than 0.001.

Narcotic addicts appear to have a mixture of both mechanisms (immune complex as well as autoimmune 7S IgG): markedly elevated platelet-bound IgG and C3C4 (2.6 and 2.4-fold classic ATP level, respectively), elevated circulating immune complexes (7.3-fold classic ATP level), anti-F(ab')2 antibodies, absence of an inverse correlation between platelet count and platelet-bound IgG. However, these patients do have specific 7S IgG anti-platelet antibody, which reacts by its F(ab')2 domain.

F(ab')2antibodies were of the IgG class and correlated with circulating immune complex level. They react with autologous, homologous patient and healthy control F(ab')2 fragments. Some anti-F(ab')2 antibodies have broad reactivity, others are more limited. Some immune complexes were shown to contain HIV antibody. It is postulated that the immune complex platelet deposition noted with homosexual and narcotic addict thrombocytopenia may in part be due to HIV antibody complexes, some of which may exist as anti-antibody complexes.
THE EFFECT OF COAGULATIOU CHANGES ON THE OUTCOME OF GASTROINTESTINAL HAEMORRHAGE
Authors: Blair S D , Tan K K , McCollum C N , Greenhalgh R M
Published: August 2018

Blood is hypercoagulable following GI haemorrhage [1], and vascular thrombosis has been reported to be the main cause of death [2]. To study the relationship between coagulation and clinical outcome, Impedance Clotting Time (ICT) wac measured daily using the Biobridge [1] and clinical outcome prospectively recorded in 125 patients with acute severe GI haemorrhage.

Mean (±se mean) ICT on admission was markedly shortened at 4.8±0.2 mins (normal range 8-12 mins) (p<0.001, t-Cest). Sixty patients received blood transfusion within 24 hours resulting in significantly prolonged ICT of 6.2±0.4 mins compared to 4.0±0.3 mins in the 56 not transfused (p<0.01). In 23 patients who rebled, the ICT at 24 hours of 6.7±0.4 mins demonstrated reduced hypercoagulability. Twenty of these 23 patients had bee:: transfused prior to rebleeding, a significantly greater proportion than in those who did not rebleed (p<0.00l). Six patients died, 3 of myocardial infarction, 1 of stroke, and 2 of continued haemorrhage. Mean ICT in the 4 patients dying from thrombotic vascular disease was 2.3±0.1 mins although 2 had also rebled.

Clinical outcome in GI haemorrhage is strongly related to coagulation changes. The main cause of death was thrombotic vascular disease.

1. Blair SD, Janvrin SB, McCollum CN, Greenhalgh RM. The effect of early blood transfusion on gastrointectinal
haemorrhage. Br J Surg 1986; 73: 792-4.

2. Allan R, Dykes P. A study of the factors influencing mortality rates fron gastrointestinal haenorrhage. Quart JMed 1976; 180: 533-50.
THROMBOGENISITY OF ENDOTHELIAL SEEDED VASCULAR GRAFT
Authors: Dryjski Jacek , Dryjski Maciej , Demuylder Charles , Janssens Th
Published: August 2018

It has recently been suggested that endothelial cell (EC) seeding of vascular grafts surfaces may lead to a less thrombo-genic surface. The present investigation was designed to study the capacity of aortic endothelium and neointima developing on seeded grafts to inactivate thrombin. In order to obtain an optimal surface for EC attachment the lyophylized porcine skin (Zenoderm®, Ethicon, Scotland) were used to form tubular grafts. Grafts seeded with enzymatically derived EC from the jugular vein was compared with control grafts that were shamed seeded with culture medium and blood. Animals with seeded grafts were divided into two groups, one group received dipyridamole treatment, theother one remained untreated. The prosthesis were implanted in the abdominal aorta in 21 dogs. Six dogs died due to different complications after surgery. The remaining 15 were sacrified 2 monthsafter graft implantations. The aorta and the grafts were excised divided into two parts, one was fixed for light and scanning electron microscopy, the otherone was analyzed for capacity to inactivate thrombin which enzymatic activity was assayed by an amiodolytic method. The grafts and aortic segments were exposed for 12 U/ml of thrombin solution. After 10 min incubation the enzyme activity decreased in the supernatant and were found in similar amounts on the grafts and aortic surfaces. Thrombin bound to endothelium was rapidly inhibited after exposure to plasma for 30 sec. In contrast, thrombin retained on the neointima of both seeded and shame seeded grafts were only minor inhibited by plasma. The morphological analyses shown that both seeded and shame seeded grafts developed a thin cellular lining covering up to 100% of the specimens’ surface area. The subcellular layer was equal in all groups. No statistical difference in morphology nor in thrombin inhibiting capacity was noted between dipyridamole treated and untreated animals. It is concluded that both the aorticendothelium and graft neointima can take up thrombin but only aortic endothelium can efficiently inactivate it. Seeding of the grafts with endothelial cellsdid not signifcantly improve its capacity to inhibit activated coagulation enzymes.
ADENOCARClNOMPrOF COLON AND THROMBOSIS: INCREASED THROMBIN,PLASMIN OR NEUTRAL PROTEASES ACTIVITY
Authors: Grand B , Blanco A , Kempfer A , Elgue G , Riveros D , Lazzari M
Published: August 2018

Thrombotic and hemorrhagic complications are common in cancer patients and are an important cause of morbidity andmortality in these subjects.Hemostatic abnormalities, with or without these complications are found in nearly 95% of these patients .These abnormalities havebeen atributed to the presence of intravascular coagulation produced by an increased fibrin gene rat ion. We report apatient with adenocarcinoma of colon and multiple venous thrombosis, whose laboratory findings were consistent with anincreased proteolytic activity different from thrombin or plasmin.

A 36 year old man with deep vein throrribosis and adenocarcinoma of colon was admitted^He started anti coagulationwith heparin and continued with acenocoimarine.In spite of a correct range of anticoagulation he developed a pulmonarthromboembolism and an axilar vein thrombosis.Coagulation studies on admissionwere as follows: Platelet count;250xl03/μl ,APTT;33 sec.,Prothrombin time;80%,FII; 150%,FV;110%,FVII-X;90%,FVIII;220%,FIX;210%,FDP;156pμ/ml.Thrombin time was shortened and clot solubility in urea 5 M was normal. Euglobulin lysis time and plasminogen(S-2251) were normal.The antithrombin 111 (AT III) concentration measured with specific antiserum (107%) and chromogenic substrates (95%) were normal.The ATIII crossed immunolectrophoresis(CIE) did not show the presence of thrombin-ATI 11 complexes .Studies on von Willebrand factor (vWF) revealed:elevation of vWF:Ag(511%) and normal ristocetin cofactor acti vity(vWF:RCof)(110%); the CIEshowed an increased mo-bi 1 ity( 1.46).Immunological levels of FXIII subunits A and S were decreased(30% and 41%) .Spontaneous proteolytic acti vity(SPA) measured with S-2288 was elevated.Though coagulation findings were consistent with consumption coagulopathy, classicaly related to thrombin gene rati on, thenormal levels of ATI 11 and the abscence of thrombin-ATIII complexes speaks against thrombin intervention.In addition, no evidence of primary fibrinolysis was observed.The increased SPA,the low levels of FXIII subunits A and S, and a faster mobility of vWF:Ag with an increased vWF:Ag/vWF:RCof ratio, suggest that hemostatic abnormalities in this patient could be due to proteolytic degradationof clotting factors by enzymes released from tumoral cells or granulocytes.
IRREVERSIBLE INHIBITION OF THE THROMBIN-MEDIATED SIGNAL TRANSFER
Authors: Visser A , Meuleman D G
Published: August 2018

The inhibition of the thrombin-mediated signal transfer by a common irreversible inhibitor Z of the factor Xa complex (Xc
a) and thrombin has been analysed for the two-step process of the Xc
a-triggered formation of thrombin andthe consecutive splitting ok a thrombin-specific substrate S. Assuming that both proteolytic processes follow simple Michaelis—Menten kinetics, that the inhibition reactions are second-order and that the prothrombin and irreversible inhibitor are in excess it can be shown that:

1. clotting time (tc) is inversely proportional to the time-averaged thrombin concentration

2. the endpoint of the conversion of the thrombin specific substrate S reached at exhaustion of thrombin and the Xc
a is inversely proportional to the square of the inhibitor concentration

3. the continously monitored thrombin generation inhibition is a more sensitive assay than the classical two-stage thrombin generation inhibition assay

4. the shift in the effective concentration range of the continuously monitored thrombin generation inhibition assay relative to the continuously monitored anti-Xa assay and to that of the continuously monitored anti-IIa assay, depends on the initial rate of formation of thrombin with the thrombin generation inhibition assay and the original enzyme concentrations of the anti-enzyme assays.

It can further be shown that the above conclusions still hold when the Z-mediated (with Z = anti thrombin III e.g.) inhibitions are potentiated by heparin(oid)s.
PHOTOMETER-LINKED MICROCOMPUTER SYSTEM ENABLES PRECISE CHROMOGENIC PROTHROMBIN TIME ASSAYS
Authors: Siekmann U , Dittrih D , Zimmermann R E
Published: August 2018

In coagulation diagnostics photometric assay procedures are in widespread use. Due to the availability of new specific chromogenic peptide substrates, automated instruments play an important role in clinical routine laboratory diagnosis. For research work, the benefit of expensive industrial photometric coagulation systems is questionable, especially as the program cannot be adapted by the user. For this purpose we developed an inexpensive microcomputer-controlled measuring system as well as a suitable photometric assay which allows to determine chromogenic clotting times with any conventional spectrophotometer.

Absorbance data were taken from the analog chart-recorder output of a double-beam spectrophotometer, digitized by a 12 bit analog-to-digital converter and read by the computer via an interface. Menu driven, user orientated / user dialogue based compiled BASIC software was written to enable data acquisition and processing.

During the chromogenic assay procedure, automatically collected absorbance data were displayed, stored, analyzed immediately, saved on disk for later kinetic analysis and printed.

Preliminary results with our chromogenic PT-assay indicate excellent reproducabi1ity of the test. The clotting time itself is defined as the interval from the beginning of the test to the moment when a preselected absorbance change occurred. Standard curves can automatically be calculated by regression routines after measurement of reference values.

It must be emphasized that the occurence of fibrinogen-generated turbidity during the chromogenic assay sometimes influences the total absorbance significantly. For this reason the reaction time has to be limited by a low optical endpoint setting.
INFLUENCE OF β-LACTAM ANTIBIOTICS ON THE PLATELETS IN VITRO EFFECTS OF SOME β-LACTAM ANTIBIOTICS ON THE BIOCHEMICAL RESPONSES OF RAT PLATELETS
Authors: Arita H , Nakano T
Published: August 2018

The inhibitory mechanism of 3-lactam antibiotics on rat platelets were studied using carbenicil1 in (CBPC) as a representative of the antibiotics. CBPC suppressed all the thrombin-induced cellular responses including shape change, secretion, and aggregation, however, it only suppressed aggregation of ADP-induced responses. This suggests that ADP binding to its own receptor was not affected by the drug while that of thrombin was inhibited. Inhibition of thrombinbinding was confirmed using 125CQSe 0f ADP-stimulated platelets, fibrinogen binding, which has an essential role for ADP-induced primary aggregation, was significantly suppressed by CBPC. Increase of a net negative charge of the membrane surface was observed after treatment of the platelets with antibiotics, and good correlation was obtained between suppression of the platelet responses and degree of net negativecharge of the antibiotics especially on the penicillin analogues. These findings strongly suggest that the inhibition of ligand binding to their own receptors is due to the increase of the negative charge of the platelet membranes which is probably caused by the antibiotic bound to the platelet membrane
EVALUATION ON EFFECTIVENESS OF LOW DOSE ASPIRIN THERAPY FOR PROTECTION OF THROMBUS FORMATION
Authors: Kariyone S , Hirakuri M , Yui T , Uchida T
Published: August 2018

In order to protect thrombus formation, administration of low dose aspirin has become common. However, its significance for this purpose is still not clear. Effect of small dose of aspirin (ASA) onplatelet aggregation and ability of malondialdehyde(MDA) formation in platelet by the addition of arachidonic acid in vitro were investigated. In clinical study, platelet aggregation, MDA formation of platelet, serum P-thromboglobulin(p-TG), platelet factor 4(PF4) and thromboxane B2(TX-B2) levelswere measured before and after low doseaspirin therapy.

Platelet aggregations by ADP, collagen, epinephrin and arachidonate as inducers were significantly suppressed under 12.5 ug/ml ASA in the medium. MDA formation of platelet was remarkably inhibited under 1.6 ug/ml of ASA in the medium in vitro.

In patients, single oral dose of 50 mg ASA showed no change on platelet aggregation while the ability of MDA formation decreased 50# of the before. Serum concentration of ASA after oral dose of 50 mg ASA only showed 1.5 to 6.0 ug/ml at maximum.

Daily dose of 50 mg ASA was continued during 10 days. Changes of various factors due to the time course were observed. Inhibitions of platelet aggregation were seen from 5th day on ADP and collagenand from 2nd day on epinephrin and arachidonate as inducers during the therapy. MDA formations of platelet were decreased quickly and it became almost zero at 5th day. ASA levels in the patient serum reached plateau as 8 or 9 ug/ml from 3rdday.

Various patients with a possibility of thrombus formation were administered daily 50 mg ASA during 10 days. Platelet aggregations and MDA production, serum β-TG, PF4 and TXB2 levels were measured before and after the therapy. Platelet aggregations by ADP, collagen and epinephrin as inducers were significantly suppressed after the therapyand MDA production of platelet was also markedly decreased after the therapy. There were no significant changes in serumβ-TG and PF4 levels while TXB2 level tended to decrease after the therapy.

From these results, it was concluded that daily 50 mg ASA oral dose was certainly effective for protection of thrombus formation.
EFFECT OF ANTIAGGREGANT ON OCCLUSION OF SAPHENOUS GRAFT CORONARY BYPASS
Authors: Lavenne-Pardonge E , Col-De Beys C , Dion R , Ponlot R , Moriau M
Published: August 2018

Double blind study on 49 patients, 24receiving aspirine-dipyri-damole, 25 a placebo. In both groups 20 patients were followed during one year. The two groupsdid not differ according to age, sex and number of coronary bypass. In all the patients, Calparin (3 x 5000 U/day) was injected subcutanously the day before andthe 7 days after surgery. In the first group dipyridamole (25 mg/ kg) was injected during the same period. The second group received a placebo IV injection. Thereafter long acting dipyridamole (400 mg/day) and aspirin (200 mg/day) were given orally in the first group, placebo in the second one. Cardiac follow-up included E.C.G. and thallium at maximum exercise. Coronarography was performed only incase of reappearence of chest pain. No difference was found between the two groups for the coagulation parameters duringthe whole year of study. Statistically significant differences between the two groups were found during the same period for β-thromboglobulin, fibrino-peptide A and the ratio Δ+ βTG/Δ+ FPA. Plateletactivity, elevated in the placebo group, was kept in normal limits in the treated group. During the two first months for the placebo and during the first month for the treated group the ratio Δ+ βTG/Δ+ FPA decreased in all patients showing the importance at this time of plasmatic hypercoagulability compared to platelet hyperactivity. During the 12 months of the study 5 thrombotic accidents (25 %) were noted in the placebo group (2 myocardial infarctions, 2 occlusions of bypass, 1 case of cerebral arterial disease) and 2 (10 % in the treated group) (1 postoperative death, 1 myocardial infarction) (NS ; p =0,21). Our results lead to two conclusions :
1) Platelet antiaggregants may influencethe permeability of saphenous .graft coronary bypass. A careful study of platelet acti-r vity with eventual change of the drug used may improve the late resultsof surgery.
2) Association of anticoagulant therapy (anti-vitamin K) during the two first months after surgery could also be useful.
DIPYRIDAMOLE INHIBITS PLATELET FUNCTION IF SUFFICIENTLY HIGH PLASMA LEVELS ARE REACHED
Authors: Kirchmaier C M , Karb O , Brickl R , Breddin H K
Published: August 2018

Dipyridamole is widely used as a platelet function inhibiting drug. It is effective in some animal thrombosis models.In healthy volunteers we studied the plasma levels after ingestion of 2 x 75 mg Dipyridamole using a fluorometric assay.We found a large variation of individualresorption rates. The highest plasma levels were detected between 30 and 60 min after intake ranging from 0,9 to 2,28 mcg/ml. Platelet retention and the percentage of stimulated platelets after addition of tissue extracts were markedly decreased. Also an increase in stypven time and slight changes in platelet volume distribution were detected during this period. To determine the plasma level of dipyridamole which is necessary to inhibitplatelet shape change and to prolong thestypven time we performed a placebo controlled double blind cross over study in 12 healthy volunteers. Blood was drawnbefore and 2 and 4 h after intake of 150 or 300 mg Dipyridamole over a p.eriod of 4 h. Using an initial dose of 37,5 mg followed by administration of 7,5 mg every15 min mean continuous plasma levels of about 0,65 mcg/ml (2 h 0,64 mcg/ml, 4 h 0,668 mcg/ml) were measured. Starting dose of 75 mg followed by multiple doses of 15 mg resulted in mean plasma levels after 2 h of 1,312 and after 4 h of 1,293mcg/ml. No side effects were observed. The plasma levels varied between the individuals. Dose-dependently a significant inhibition (p 0,01) of tissue extract induced platelet stimulation and a prolongation of stypven time was observed. The antithrombotic effect of Dipyridamole is not correlated with the inhibition of platelet aggregation, but may be closely related to the inhibition of platelet adhesion and platelet factor 3 avai1abi1ity.
AN EXTRACT OP FEVERFEW INHIBITS INTERACTION OP HUMAN PLATELETS WITH COLLAGEN SUBSTRATES
Authors: Lösche W , Mazurov A V , Groenewegen W A , Heptinstall S A , Repin V S
Published: August 2018

Feverfew (Tanacetum parthenium) has been used since ancient times as a herbal remedy for migraine, fever and arthritis. Recently it has been shown that extracts of feverfew inhibit aggregatory and secretory responses in human platelets induced by various soluble agonists.

The interaction of platelets with surfaces coated with human collagens of type III (C III) and IV (C IV) has been studied by measuring the deposition of 51-Cr-labelled platelets and by scanning electron microscopy. Experiments were performed using platelet-rich plasma (PRP) and suspensions of gel-filtered platelets. Platelets were deposited on C III mostly as surface-bound aggregates. In contrast they were deposited on C IV mostly as spread forms of individual cells. Formation of aggregates on C III was more extensive for PRP than for GPP; in contrast platelet spreading on C IV was more extensive for GPP than for PRP.

Feverfew extract inhibited the deposition of 51-Cr-labelled platelets on both C III and C IV in a dose dependent way. Similar concentrations of extract were needed to inhibit the formation of surface-bound aggregates and to inhibit platelet spreading in both PRP and GPP.

The results indicate that feverfew may have antithrombotic potential in addition to its claimed benefit in certain clinicalconditions.
INFLUENCE OF CELL CYCLING ON MAGNITUDE OF RESPONSE OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS (EC) TO NOREPINEPHRINE (NE) AND HISTAMINE (H)
Authors: Gupta M , Stewart G J
Published: August 2018

EC have several roles in preventing thrombosis. Efficiency with which EC fulfill these might well influence outcome of potentially thrombotic stimuli. Thus, factors that influence EC function are of interest. As a general measure of EC response to NE and H the level of cAMP was measured. EC were harvested and passaged by enzymatic digestion and grown in DMEM plus 10% fetal calf serum and antibiotics. cAMP was assayed by radioimmunoassay. Percentage cycling cells was determined by autoradiography of cultures pulse labeled with tritiated thymidine and was increased by hydroxyurea treatment.

Basal cAMP was remarkably consistent (less than 10% difference) for cells isolated from different cords, studied after different number of passages, fresh or frozen and with varying percentage of cells cycling. However, magnitude of response to agonists was highly variable. Within the same cell lot between 5-8 passages, magnitude of response was directly proportional to percentage of cycling cells but magnitude ofresponse for different cell lots varied over several fold.

EC response to NE was mediated through B-adrenergic receptors and that to H through H receptors. Variability might be caused by differences in receptor number or some intracellular process.
EFFECTS OF MECHANICAL FORCE ON BLOOD FIBRINOLYTIC ACTIVITY
Authors: Takashima N , Tsuruta H , Higashi T , Watanabe M , Isomoto A , Tyuma I
Published: August 2018

The effects of exposure to mechanical force on the blood and blood vessels were studied concentrating on the factors of thrombosis and hemostasis. (I) The palms of 10 healthy males were exposed to mechanical vibration (35 Hz, 5 G, 5 mm p-p) for 5 min. Euglobulin fractions were separated from the pre- and postvibration blood samples. The fibrinolytic activity, measured with the fibrin plate method, increased after vibration; fibrinolytic areas (pre/post) = 15.3 ± 6.0/18.6 ± 8.4 mm2 . The tissue-type plasminogen activator (t-PA) from endothelial cells of blood vessels was measured with the same blood samples by an enzyme-linked immunosorbent assay using monoclonal antibodies. It was proportional to the fibrinolytic activity; t-PA (pre/post) = 1.36 ± 1.09/1.58 ± 1.21 ng/ml. This suggests that the t-PA from the endothelial cells participates in increasing the fibrinolytic activity. (II) Six healthy males performed the side-step test for 5 min. At pre- and post-exercise, blood was collected from the great saphenous vein and its t-PA was measured to investigate the direct effect of mechanical force on the sole of the feet. As a control, the t-PA of the median venous blood was also measured. When the side-step test was performed with bare feet, the t-PA increased in the great saphenous venous blood, but not so much in the control. On the other hand, when the same test was done with the subjects wearing shoes, the t-PA did not increase. These data show that the mechanical force directly releases t-PA from the endothelial cells of the foot vessels in the soles of the feet, and that shoes can protect the feet from shock.

The influence of physical exercise on fibrinolytic activity is well known. In the mechanism, endothelial cells are thought to be stimulated by vasoactive hormones. However, the direct influence of mechanical force on the blood vessels must also be considered.