bluuebunny/crossref_metadata_2025 huggingface dataset:
THE "LUPUS ANTICOAGULANT" INDUCES FUNCTIONAL CHANGES IN ENDOTHELIAL CELLS AND PLATELETS
Authors: Schorer Anna E , Watson Kathleen V
Published: August 2018

The presence of the "lupus anticoagulant" (LA) predicts a clinical syndrome of excessive arterial, venous and microvascu-lar thrombosis. LA is an antibody which reacts with negatively charged phospholipid (PL) species in vitro. Since PL is involved in many aspects of the regulation of thrombosis, we postulated that LA might modify one or more of the membrane-(PL-dependent reactions of platelets and endothelial cells (EC). Blood samples from 20 patients with a history of thrombosis were tested for the presence of LA (kaolin PTT) and titres determined. LA-positive (LA+) sera and plasma were compared to LA-negative (LA−) samples from normal donors (n=6) or patients who had lupus but no clinical thrombosis (n=4). These specimens were tested in a panel of assays. The thrombin-stimulated release of prostacyclin (PG12) from cultured human EC was markedly reduced (52%±12.5 s.e.) by preincubation of the EC with LA+ sera (30 minutes). Purified LA+ IgG from one patient reproduced this effect. Thrombin induction of EC synthesis of the procoagulant, tissue factor-which is dissociable from prostaglandin metabolism-was also inhibited by LA+ sera. Normal platelets incubated in LA+ plasma became refractory to thrombin (1 unit/ml) but retained their responsiveness to epinephrine and ADP. The reduced responsiveness to thrombin was not due to altered (specific or total) binding of thrombin. The cleavage of Factor X by Factor VII requires PL as a co-factor for the EC procoagulant, tissue factor (TF). Unlike the inhibitory effect of LA on thrombin activation of EC and platelets, this distinct membrane-(PL-) dependent function was variably enhanced by LA+ sera. Brief (20 min) exposure of EC to LA+ sera increased TF co-catalysis of Factor VII cleavage of Factor X (measured by chromogenic Xa substrate, S-2222) by up to 10 fold (p<0.05, unpaired t test). This effect was not the result of EC disruption or changes in whole-cell TF content. These data suggest multiple, complex and heterogenous effects of LA, including impaired production of PG12, impaired EC modulation, and heightened ability of endogenous EC tissue factor to initiate coagulation. These (and perhaps other) membrane-dependent effects may contribute to the tendency of LA+ patients to develop clots.
HETEROGENEOUS REACTIVITY OF HUMAN MONOCLONAL ANTIPHOSPHOLIPID ANTIBODIES (HMA) WITH ENDOTHELIAL CELLS (EC), PLATELETS AND DNA
Authors: IZAGUIRRE C A , DUFF S , HASHMENT S , SMITH C D , MENG Q H , RAUGH J
Published: August 2018

Antibodies that react with cardiolipin (ACL) and/or have lupus anticoagulant (LA) properties are implicated in the thrombotic diathesis of patients with the antiphospholipid antibody syndromes: recurrent venousand arterial thrombosis, fetal wastage, thrombocytopenia and CNS disease, often associated with systemic lupus erythematosus. We studied a panel of 16 HMA with LA and/or ACL properties (J Biol Chem 261:9672 1986) for their reactivity with platelets (ELISA),DNA (ELISA), with surface antigensonEC (EC-ELISA) and with whole cell EC extracts as detected by western blot and immunoperoxidase (EC-WB). Three control HMA werenonreactive in all assays.

Five HMA with LA activity alone did not react with EC. Five of 6 HMA-ACL reacted with EC both on ELISA and WB. All of the HMA-ACL also reacted with DNA and platelets but 4 did not react as LA. Our findings indicate that HMA antiphospholipid antibodies(LA and ACL) appear to be heterogeneous in terms of their spectrum of reactivities, thus, the ACL and LA tests may in some cases recognize antibodies with different properties and perhaps different pathogeneticmechanisms.xs
DISSEMINATED INTRAVASCULAR COAGULATION (DIC) AND ACUTE LEUKEMIA:IDENTIFICATION OF A NEW CELLULAR PROCOAGULANT
Authors: Falang A , Alessio G M , Donati M , Barbui T A
Published: August 2018

There is an enhanced incidence (>50%) of severe coagulopathy in association with several types of acute leukemias. Cell associated procoagulants are considered important in this context. So far only a Tissue Factor (TF)-type procoagulant has been described in leukemic cells. We have set up here the experimentalconditions to identify other possible cellular procoagulants in leukemia. We have tested blast cell extracts from 21 patients with 5 different cytological subtypes (from Ml to M5 of acute non lymphoid leukemia (ANLL), according to theFAB classification, in order to assay whether they express "cancer procoagulant" (CP), a F VH-independent FX activating cysteine proteinase (Falanga … Gordon, 1985; Donati, et al. 1986). All the samples shortened the recalcification time of normal human plasma, the effect being significantly greater (p<0.001) in the M3 group. The activity was 20% to 100% independent from the presence of FVII and was susceptible to 2 cysteine proteinase inhibitors (Iodoacetamide, 2 mM, and HgCl2 ,0.1 mM) in all of the extracts but the M5 type. In addition, M2 and M3 samples directly activated pure FX in a two stage clotting assay. Control cell extracts from 10 healthy donors did not show any procoagulant activity, under the same conditions. This study provides evidence for a new procoagulant expressed by cells of ANLL; the peculiar characteristics of this procoagulant (i.e. its confinement to the malignant phenotype, its shedding into the plasma, its possible modulation by vitamin K antagonists) make this observation of potential interest in the development of new diagnostic and therapeutic tools in ANLL.
THE DISTRIBUTION OF CROSS-LINKED FIBRIN IN HUMAN LUNG CARCINOMA PARALLELS THAT OF ACTIVATED HOST MONONUCLEAR LEUKOCYTES: IMMUNO-HISTOLOGIC STUDIES WITH MONOCLONAL ANTIBODIES
Authors: Rickles F R , Hancock W W , Kobzik K , Hogg N , O’Hara C
Published: August 2018

Previous immunohistologic studies of human lung carcinoma, using polyclonal antibodies to antigens shared between fibrinogen (FGN) and fibrin (FB), showed that FGN/FB were associated withtumor cells. These findings were interpreted as evidence of the presence of tumor-associated procoagulant activity(PCA). Wecompared the distribution of coagulation-associated proteins in 16 casesof human carcinoma of the lungofvarying histologic types, using polyclonal antibodies to FGN/FB and monoclonal antibodies (mAb) to cross-linked fibrin (XL-FB;UC-45), fibronectin (FN;PHM13), factor VIII (vWF:Ag)and a tissue factor-related antigen(TF:RAg;A1 -3)- Host mononuclear leukocytes were identifiedusing various mAb toT cells and macrophages, and studied for their expression of receptorsfor interleukin-2 (IL-2R). Positive resultsare summarized

In addition, studies of the mononuclearcells adjacent to tumors in 12/12 casesshowed the presence of tumor-associated macrophages, 10/11 showed T cells,mainly T8+, and A/5 showed corresponding expression of IL-2R, suggesting cell activation.

The use of highly specific mAb showed that XL-FB is actually more selectively distributed than is found using polyclonal antisera to FGN/FB, and indeed XL-FB was largely confined to those areas adjacent to tumors which are rich in mononuclearcells. These findings suggest that fibrin deposits in human carcinomasof the lung may be due todevelopment of PCA by activated host mononuclear cells, rather than tumor cells.This lack of XL-FB on tumor cellsinspite of A1- 3 binding suggests that TF:RAg may not be available on the tumor cell surface for the activation of clotting. Further studies are neededto define the functional capacity of PCA molecules on tumor cells and tumor-associated mononuclear cells in situ.
COAGULATION ACTIVATION AND OCCURRENCE OF HYPOFIBRINOGENEMIA IN PATIENTS WITH ACUTE NONLYMPHOBLASTIC LEUKEMIA
Authors: Pabinger I , Lechner K T , Bettelheim P , Dudczak R , Hinterberger W , Korninger C , Medicine I Int
Published: August 2018

122 consecutive adult patients (pts) with acute nonlympho-blastic leukemia (ANLL) were studied for evidence of coagulation activation andoccurrence of hypofibrinogenemia. In 16 pts (13%) decreased fibrinogenlevels (< 1.5g/l) were found.Data on fibrinopeptide A levels (FPA), ethanol gelation test (EGT) andfibrin(ogen) degredation products(FDP) are listed in the table:

The occurrence of hypofibrinogenemia was not correlated to the peripheral blast cell count. 10/18 pts with M3 (FAB-classifica-tion), 2/46pts with M2, 2/25 pts with M4 and 2/14 pts with M5 leukemia developed hypofibrinogenemia. Early death occurred in 31% of pts with hypofibrinogenemia (in most pts due to hemorrhage) and only in 10% without (in most pts due to septicemia). No significant difference in remission duration was found between the 2 groups. 48 patients were reinvestigated at the time of recurrence of disease. 5 of 7 patients with and 38 of 41 patients without initial hypofibrinogenemia had a coagulation pattern similar to the one observed at first presentation.

The following conclusions can bedrawn:(1) In most patients with ANLL there is evidence of coagulation activation. (2) Hypofibrinogenemia in patients with ANLL appears to bethrombin mediated. Therefore the term disseminated intravascular coagulation (DIC) may be used. (3) DIC is strongly associated with the subtype M3 (promyelocytic L), but may as well occur in other ANLL subtypes.(4) The presence of DIC seems to be a specific property of certain leukemic clones. This assumption is supported by the coagulation studiesduring recurrence of ANLL. (5) Patients with low fibrinogen levels areat high risk of early death due to hemorrhagic complications.
THROMBIN-ANTITHROMBIN III COMPLEX - A NEW PARAMETER FOR DETECTION OF THROMBOTIC STATES
Authors: Pelzer H , Egbring R , Seitz R , Blanks H B , Cazzola M , Heimburger N
Published: August 2018

Recently, we have developed an enzyme-linked immunosorbent assay (ELISA) for determination of thrombin-antithrombin III complex (TAT) in numan plasma (1). The test system follows the sandwich principle and uses two different antibodies directed against human thrombin and human antithrombin III, respectively. The antibodies bind selectively to the corresponding antigen moieties of TAT. The assay was calibrated with definite concentrations (2.0 to 60 μg/l) of preformed purified TAT added to TAT-poor plasma. Plots of absorbance 492 nm against TAT concentration revealed a linear correlation (r = 0.98). The lower limit of sensitivity of the assay was 0.5 μg/l. Mean coefficients of variation of 4 % (intraassay) and 7.5 % (interassay) were found for TAT concentrations between 2 and 60 μg/l. A reference range from 0.85 to 3.0 μg/l was calculated from TAT concentration in plasma samples from 88 healthy donors (mean value ± SD: 1.45 ± 0.4 μg/l). In plasma samples from patients with pulmonary embolism confirmed by lung-scan (n = 17), TAT concentrations between 10 and 20 μg/l were measured. In patients with deep vein thrombosis confirmed by phlebography (n = 15), TAT were found up to 7 - 13 μg/l. Patients with septicaemia associated with a consumption coagulopathy (n = 10), and patients with acute hepatic failure (n = 5) showed markedly increased TAT values. In plasma samples from patients with various metastatic malignant diseases, TAT concentrations of 30 to 60 μg/l could be measured. From these data we conclude that measurement of TAT can be a sensitive parameter for specific detection of a latent activation of the clotting pathway.
Increase of Thrombin-Antithrombin III (TAT) Complex Plasma Levels in Thromboembolic Diseases during Thrombolysis
Authors: Seitz R , Pratorius G , Blanke R , Strauer B B
Published: August 2018

Recently an enzyme immuno assay of thrombin-antithrombinlll complex (TAT) plasma levels was developed by PELZER et al. (Thromb. Haemost. 54:24,1985). This test appears to be useful in the detection of intravasal thrombin generation, since all of 17 patients (pts.) with pulmonary embolism and 15 of 16 pts. with deep vein thrombosis (DVT) showed elevated values above 3 ng/ml.

In 9 pts. with acute myocardial infarction (AMI) the TAT levels increased significantly (p 0.001) 3 to 6 hours after thrombolytic therapy with 1.5 million units streptokinase (SK) over 30 minutes. A concomitant increase of fibrinopeptide A (FPA) levels (p=0.048) was observed. In contrast, 8 AMI pts. treated with heparin showed an insignificant increase of TAT and FPA. In 7 DVT pts. the TAT levels rose significantly (p 0.001) within 6 hours after start of urokinase (UK) infusion, while the FPA levels were enhanced prior to treatment and showed no further increase.

In order to assess the in vitro effects of SK and UK on TAT levels, clots obtained by recalcification of citrated plasma were incubated in heparin (2 units/ml) plasma. An increase of TAT occurred after addition of SK or UK, which was less pronounced when the clots were rinsed extensivly or squeezed before incubation. When SK or UK were added to plasma in the absence of a clot, still a small increase of TAT occurred which was absent in saline controls.

The data suggest that SK and UK action is associated with the generation of TAT complexes. In vivo, thrombin or thromboplastic material might be released by enhanced "wash out" from the recanalized coronary artery or from the reperfused in-farcted myocardium. Thrombin might also be released from binding sites on fibrin clots or fibrinogen. It is conceivable that these findings contribute to the understanding of reocclusion of infarct vessels after thrombolytic therapy. This points to the importance of careful anticoagulation in patients receiving thrombolytic therapy.
THE PHYSIOLOGICAL ANTITHROMBIN (BETA) VARIANT WITH INCREASED HEPARIN AFFINITY LACKS CARBOHYDRATE AT ASN 135
Authors: Brennan S O , George P M , Carrel R W , Jordan R
Published: August 2018

Some 10% of the antithrombin fromnormal human plasma can be isolated onthe basis of its higher affinity forheparin Sepharose. Not only does this ATIII beta have a higher heparinaffinity, but it is also less negatively charged and has a lower molecular weight than normal AT-III alpha.Neuraminidase and Endo F treatment suggest the loss of a single carbohydrate sidechain from beta AT-III (Peterson and Blackburn. J Biol Chem 1985,260, 610)

Antithrombin has a single polypeptide chain with biantennary carbohydrate attachment sites at asparagines 96, 135, 155 and 172. The 25 kDA CNBr fragment of AT-III alpha (residues 104-251) runs at 22.5kDa in the case of AT-III beta yet they have the same N and C-terminal sequence. Tryptic peptide maps of theseCNBr fragments showed that a single neutral (glyco) peptide, Lys-Ala-Asn⋆-Lys was missing from AT-III beta and replaced by twobasic peptides, Lys, Ala, Asn, Lys and Ala, Asn, Lys; indicating the absence of an oligosaccharide sidechainon asparagine 135. This was confirmed by chromatography of tryptic peptides on Con A-Sepharose followed by mapping of the bound and unbound fractions. There were two new unbound peptides (Lys, Ala, Asn, Lys and Ala, Asn, Lys) in AT-III beta. The corresponding peptide Lys-Ala-Asn⋆-Lys was the only peptide missing from the bound glycopeptide fraction.

The absence of carbohydrate at Asn 135 explains the increased heparinaffinity of beta AT-III as on the molecular model this oligosaccharide forms a negative and bulky shell on the perimeter of the positive bindingsite centred on Arg 47.Whether the loss of the sidechain is due to circulatory removal is not known but a recent variant identification confirmsthat it may alternatively be due to failure of attachment at the time ofsynthesis. Potentially the loss or modification of oligosaccharide 135 provides another level of control of coagulation by altering the avidity of AT-III for thrombin when heparin activation is suboptimal.
EVIDENCE FOR SECONDARY SITES OF HEPARIN BINDING TO ANTITHROMBIN III (AT) ISOFORMS
Authors: Carlson Timothy H
Published: August 2018

AT exists as two isoforms differing in affinity for heparin (Thromb Res 27:23,1982; JBC 260:610,1985). The binding of human AT isoforms to commercial heparin, or purified unbleached heparin was studied. These heterogeneous, mixed-affinity (MA) heparins were coupled to agarose. When columns of these resins were eluted with NaCl gradients, after mixtures of the higher (ATvh) and lower (ATh) affinity isoforms of AT had been applied, ATvh always eluted after ATh. To determine the nature of this difference, low-affinity (LA) heparin was prepared by repeated passage of MA heparin over an ATh-ConA-Sepharose column. Neither AT isoform bound during passage over a LA-heparin agarose column equilibrated with 0.15 M NaCl. However, the ATvh was significantly retarded, while ATh eluted at about the same position as BSA. To demonstrate that ATh also interacts with a LA-site on heparin, a column containing MA heparin was loaded with ATh until the effluent concentration reached a steady state. Under the conditions, 21 mg of ATh was associated with the column in the steady state. Of this 10 mg eluted during a wash with 0.15 M NaCl, and 11 mg with 1.5 M NaCl elution. When the same experiment was performed using ATvh in place of ATh, approximately 25% more ATvh bound in both the steady state and to the washed column. To demonstrate that a secondary interaction is responsible for the separation of ATvh and ATh during heparin-agarose chromatography, the isoforms were applied together to MA-heparin columns in less than saturating amounts. When NaCl gradients were used to elute the columns in a reverse direction (bottom to top of column), ATh and ATvh co-eluted at relatively low salt concentrations. Thus, separation results during filtration of ATh and ATvh through the columns, after neutralization of the HA interaction. Finally, to determine if ATh and ATvh compete for the same binding sites on heparin, a MA-heparin column was loaded with a saturating amount of ATh. When ATvh was applied to this column, ATh was quantitatively displaced. It is concluded that (1) both AT isoforms bind LA sites on heparin (2) heparin contains many more LA sites than the HA ones; (3) ATvh binds more strongly to LA sites than ATh; and (4) LA binding may enhance activation of AT at the HA site by increasing the rate at which AT binds to this site.
DEEP VEIN THROMBOSIS PROPHYLAXIS IN SURGICALLY TREATED FRACTURED HIP PATIENTS
Authors: Powers P J , Gent M , Jay R , Hirsh J , Levine M , Turpie G
Published: August 2018

Deep vein thrombosis is a major complication in'patients treated surgically for fractured hip. Methods employed toreduce the risk of thrombosis include dextran, ASA, warfarin, low or adjusted dose heparin and calf compression, but none has widespread acceptance.A randomized trial wascarried out to assess the effectiveness of sodium warfarinand acetyl salicylic acid(aspirin) compared to placebo inthe prevention of venous thrombosis in fractured hip patients. One hundred and ninty four patients were randomizedto receive warfarin (65 patients), ASA (66 patients) or placebo (63 patients).Prophylaxis commenced post operatively and continued for 21 days or until discharge, if earlier.Warfarin patients received 10 mg sodium warfarin orally as soon as possible after surgery. Warfarin was then given daily according to the prothrombin time (PT), to obtain a PT of 16 seconds on the 5th post operative day. The PT was maintain at 16 to 18 seconds until the end of treatment.ASA and placebo patients received enteric coated tablets, 650 mg twice daily, in a double blind fashion beginning as soon as possible post operatively and continuingto the end of treatment. Surveillance testing and I-fibrinogen leg scanning and impedance plethysmography was performed and venography was done if either test suggested thrombus at the popliteal vein or above. Otherwise venography was performed at day 21 or prior to discharge, if earlier. Venous thrombosis occurred in 13 patients (20%) in the warfarin group, 27 patients (^0.9%) in the ASA group, and 29 patients (46%) in the placebo group (P=0.005). Proximal vein thrombosis or pulmonary embolism occurred in 6patients (9.2%) in the warfarin group,7 patients (10.6%) in the ASA and 19 patients (30.2%) in the placebo group (P=0.002). Two major hemorrhages occurred in the warfarin group, none in the ASA group, and 2 in the placebo group.The results of this study show sodium warfarin to be safeand effective in reducing thromboembolic complications infractured hip patients and ASA to be effective in reducing thrombosis involving the proximal deep veins of the lower limbs in these patients.
ENDOGENOUS VASOPRESSIN RELEASE CONTRIBUTES TO THE THROMBOTIC RISK OF HIP SURGERY
Authors: Wilson J , Grant P J , Boothby M , Davis J A , Prentice C R M
Published: August 2018

Vasopressin (aVP) infusions in man that simulate physiological concentrations in plasma produce increases inboth factor VIII and plasminogen activator activity (PAA) and we have found evidence that aVP release contributes to the activation of coagulation during abdominal surgery. The aimof this study was to investigate whether aVP has a similar role in theregulation of haemostasis during hipsurgery. Venous blood samples were taken for FVIII:C, FVIII RiCof, vWF:Ag,fibrinopeptide A (FPA), ECLT, platelet aggregation in whole blood andaVPfrom separate venepuncture sites in 7 patients undergoing elective hip surgery. Samples were taken pre-operatively, post induction of anaesthesia, at skin incision, during divisionof the femoral neck, reaming of the acetabulum, cementing of the prosthesisand on thefirst post-operative day. FVIII:C increased during the operation from a geometric mean of 70%pre-operativelyto 109% (p < 0.05) post-operativgly.vWF:Ag and FVIII RiCof rose in a similar manner. PAA (106 /ECLT2) rose significantly from 12 units pre-operatively to a peak value of 167 units(p< 0.001) at prosthesis cementing,andpost-operatively fell to subnoral levels. FPA concentrations followed a similar pattern rising from13 pmol/ml to 58 psmol/ml (p < 0.02)atprosthesis cementing, and falling to9pmol/ml post-operatively. PlasmaaVP rosefrom 0.5pg/ml pre-operatively to 40pg/ml (p < 0.01) at division of the femoral neck, and returned to0.5 pg/ml post-operatively. There were no changes in platelet aggregation in whole blood using a single doseof 1.5 μM ADP. These results are similarto those we have observed duringabdominal surgery. They confirm thaduring surgery, thrombin generation occurs with increased PAA,both of which are preceded by a risein aVP. This isconsistent with the hypothesisthat aVP is an important mediatorof changes in haemostatic function occurringduring surgery, and we are now investigating the relationship between intra-operative changesin haemostaticfunctionand risk of post-operative DVT.
ARG-GLY-ASP (RGD) PEPTIDES INCREASE THE EXPOSURE OF THE CARBOXYL TERMINAL REGION OF THE HEAVY CHAIN OF GPIIB ON THE PLATELET SURFACE
Authors: Lam S , Plow E F , Frelinger III A L , Smith M A , Loftus J C , Ginsberg M H
Published: August 2018

RGD peptides and those derived from the fibrinogen γ chain (e.g., γ 400-411 (H12)) are inhibitors of platelet aggregation and fibrinogen binding. These two sets of peptides interact with a common adhesive protein receptor on platelets which contains GPIIb-IIIa (JBC, 262:947, 1987). A 17 amino acid sequence near the carboxyl terminus of GPIIbα recognized by the PMI-1 monoclonal antibody (JCI, 78:1103, 1986), isexpressed on the cell surface under certain conditions which are not permissive for fibrinogen binding. Thus, achange in the surface orientation of GPIIb is associated with inhibitionofplatelet aggregation and fibrinogen binding. To investigate whether or notsuch changes in GPIIb-IIIa occur during peptide inhibition of plateletfunction, we examined peptide effectson exposure of the PMI-1 epitope. Three different RGD peptides induced reversibly increased binding of 125I-PMI-1 to both resting and thrombin-stimulated platelets. Specificity of this effect was demonstrated bythe failure of the inactive peptides GRGESP andSDGR to induce PMI-1 binding. This effect does not appear to requireviable platelets since it was observed on fixed cells and in detergent solutioncontaining purified GPIIb-IIIa. Theγchair, peptides provoked asimilar effect, but required greater concentrations than the RGD peptides.These results indicate that RGDpeptides, in addition to their presumed competitive effect on adhesive protein binding to platelets, are also capableof increasing the surface exposureofthe carboxyl terminus of the heavy chain of GPIIb, thus potentially altering platelet function.
INHERITED DEFICIENCIESCAN AFFECT SEPARATELY THE PLATELET MEMBRANE GLYCOPROTEIN Ilb-IIIa COMPLEX AND THE LEUKOCYTE LFA-1, Mac-1 and pl50,95 COMPLEXES
Authors: Pidard D , Fischer A , Bouillot C , Ledeist F , Nurden A T
Published: August 2018

The human platelet membrane glycoprotein (GP) IIb-IIIa complex and a family of functional leukocyte cell membrane antigens, LFA-1 (L), Mac-1 (M) andpl50,95 (X), possess knownstructural analogies. Similaritiesinclude a heterodimeric structure with a high mol. wt. αsubunit (Mr∽ 145-180 kDa) , associated nonconvalently with a lower mol. wt.β-subunit (Mr ∽ 90-95 kDa),anda partial amino acid sequence hommology between GP lib and αL or CKM. Furthermore, GP lib, α L and αM were reported to be co-expressed in murine cells transfected with a 20 kilobase human DNA fragment.

To address the question of a possible genomic linkage between these related glycoproteins of different cells, we have examined patients with either ‘LFA-1 immunodeficiency disease’ or with Glanzmann's thrombasthenia (GT). S.Bo. and P.Ce. are children affected by recurrent bacterial infections since birth. Cytofluorimetric analysis using monoclonal antibodies (MAbs) for the αL, αM,αX and β subunits demonstrated that the surface expression of LFA-1, Mac-1 and pl50,95 intheir leukocytes was ≤1% of the normal values. Platelets from both patients aggregated normally and readily bound the MAbs AP-2 (anti-GP IIb-IIIa) or Tab (anti-GP IIb). Crossed immunoelectrophoresis confirmed the presenceof GP IIb-IIIa complexes, whileSDS-polyacrylamide gel electrophoresis showed a normal migration of GP IIb and GP IIIa. Patients C. Bl. and M.Ca.are typical of the type I subgroupofGT. Their platelets are unable toaggregate and contain < 1% of the normal platelet content of GP liband GP Ilia.Cytofluoro-graphy showed a normal expression of LFA-1, Mac-1 and 150,95 on the surface of lymphocytes, polymorphonuclear cells and/or monocytes of both patients. Our data thus show that despite sequence homologies and a potential genomic linkage, the cellular expression of the platelet GP IIb-IIIa complex and of the LFA-1 related leukocyte antigens may be dissociated in genetic disorders where anabnormal glycoprotein distribution may be restricted to one type of cell.
STUDIES ON THE INVOLVEMENTOF GPIIb/IIIa COMPLEX IN PLATELET AGGREGATION WITH TWO MONOCLONAL ANTIBODIES
Authors: Ryckewaert J J , Valiron O , Newton I A , Concord E , Prenant M , Berthier E
Published: August 2018

The involvement of the glycoproteins IIb/IIIa in the binding of fibrinogen to stimulated platelets and in the aggregation of these cells is well documented. Monoclonal antibodies (MoAb) directedagainst these two glycoproteins are useful probes in functional studies. Two MoAbs have been obtained against thetwo glycoproteins in their heterodimer form. Both MoAbs immunoprecipitate the complex from platelet lysates and only bind to platelets if the complex of the two glycoproteins is not dissociated.Studies onthe effect of the two MoAbs on platelet function have been performed. Firstly CS9,a murine IgG 2a, inhibited the binding of fibrinogen to stimulated platelets and prevented platelet aggregation. Fab CS9 obtained bypapain digestion had a similar activity. The binding of I125 Fab CS9 to resting platelets was similar to that of ADP-stimulated platelets (30 000 MoAbs/platelet). Similar behaviour of MoAbs against GPIIb/IIIa has been repeatedly reported and is considered as evidence that the fibrinogen receptor is the GPIIb/IIIa complex. The second MoAb (CS3),an IgGJ, subtype had a different action on platelet function.Contrary to all other known antibodies directed against the GPIIb/IIIa complex, CS3 or (Fab’2)CS3 induced the bindingof fibrinogen to platelet in the absence ofany plateletstimuli. Washed platelets in the presenceof CS3 underwent immediate and extensive aggregation, even in the absenceof fibrinogen. The stimulatory activity of MoAb CS3 on platelet can be eliminated by PGEJ. Monovalent CS3 (Fab fragment) failed to exhibit the properties of the parent antibody and had no effect on platelet function. The most likely explanation of the action of CS3 on platelet function may be that crosslinking of two GPIIb/IIIa complex mediated by the antibody leads to platelet stimulation. Whetheror not this mechanism is evocative of a physiological event when platelets are stimulated remains to be demonstrated. When both MoAbs are used in combination, the binding of fibrinogen and platelet aggregation are prevented by CS9 despite the stimulatory activity of CS3. Thus both MoAbsareable to bind to the GPIIb/IIIa complexat the same time.
HUMAN cDNA CLONES FOR THROMBOPLASTIN
Authors: Bjorklid E , Johansen T , Pendurthi U R , Rao L V M , Warn-Cramer B , Rapaport S T
Published: August 2018

A rabbit polyclonal antibody which was monospecific for thromboplastin (TP) apoprotein from human brain according to a number of criteria was used to screen two human placenta cDNA libraries in the expression vector λgt11, one randomly primed and one oligo-dT primed, by the method of Young and Davis. 23 positive clones expressing TP related antigen were isolated and plaque purified. DNA from the different clones was isolated and the TPcDNA inserts released by EcoRl digestion. The inserts could be classified into11 size classes ranging from approx. 300-1100 base pairs.The largest insert (1100 bp) was subcloned intothe plasmid vector pGEM-1. When the nick-translated plasmid (pTP4-l) was used as a probe to screen the phage clones by slot blot hybridization all the 23clones hybridized to the 1100 bp insert. A λgt11TP4 lysogen expressed β-galactosidase- TP4 fusion peptide upon IPTG induction as shown by immunobinging studied using two different antibodies to TP apoprotein: the rabbit antibody originally used to screen the libraries and an antibody raised in goat against human brain TP purified by affinity chromatography on a Factor VII-antiVII-agarose column.
STRUCTURE AND PROPERTIES OF THE HUMAN TISSUE FACTOR APOPROTEIN
Authors: Morrissey J H , Fair D S , Edgington T S
Published: August 2018

Tissue factor (TF), an integral membrane glycoprotein, is an initiating molecule for the coagulation protease cascade. TF must reside in a phospholipid membrane for optimal activity where it functions as the receptor and essential allosteric activator for factor Vll/VIIa.TF apoprotein was purified from human brain and placenta using factor Vll-affinity chromatography or immunoaffinity chromatography with a mouse anti-TF monoclonal antibody. Both methods resulted in a homogeneous preparation consisting of a highly glycosylated 47 kDa heavy chain and a 12.5 kDa light chain.Removal of asparagine-linked carbohydrate chains with glycopeptidase F reduced the apparentmolecular weight of the heavy chain to 37 kDa but hadno effect on the mobility of the light chain in SDS gel electrophoresis. Electrophoretic analysis of theintact protein with and without reduction indicates that the light chain is disulfide-linked to the heavychain in about half of the TF molecules and is notessential for function.The majority of polyclonal andtwenty- nine monoclonal antibodies against purified TF strongly inhibit coagulation and in all cases aredirected against epitopes on the heavy chain alone. Functional regions of the TF heavy chain have been investigated using a library of twenty-nine monoclonalantibodies and a series of overlapping, synthetic oligopeptides based on sequence information obtained from cloning the cDNA for TF. Supported by NIH grantsHL-16411 and CA-41085.
DIFFERENTIAL EXPRESSION AND SUBCELLULAR LOCALIZATION OF TISSUE FACTORIN A CONSTITUTIVE VERSUS AN INDUCIBLE CELL TYPE
Authors: Morrissey J H , Gregory S A , Edgington T S
Published: August 2018

Tissue factor (TF) is an integral membraneglycoprotein and receptor present on a variety of cells outside of the vasculature, but normally absent from intravascular cells. TF plays a central role in initiation of coagulation by rapidly binding and allosterically activating bound factor Vll/VIIa, which proteoly-tically activates coagulation factors IX and X. This protease cascade appears to play a role in the cellular inflammatory response, during which endothelial cells and monocytes/macrophages can be induced to express cell surface TF.Monocyte TF can be induced in response to endotoxin and also via direct interaction with activated T cells and by a specific lymphokine.

We have developed a panel of polyclonaland twenty-nine high affinity monoclonal antibodies to human TF. The antibodies recognize TF epitopes under a broad range of conditions, some of which rapidly and efficiently neutralize <95% of TF activity isolated from brain, placenta and expressed bycultured cells. Using these antibodies in immunohistochemical assays, we haveobserved little or no TF antigen cytologically associated with resting monocytes, noTF activity, and following stimulation, the cytologic appearance of TF antigen parallels the acquisition of TF activity.Immunohistochemical staining of stimulated monocytesis diffuse, consistent with homogeneous cell-surface distribution of the TF molecule.In addition, the normal human fibroblastic cell lines GM1380 and GM1381, whichexpress TF const itutively, show a cytologically different and much more intense pattern of intracellular inclusions of TF. This is consistent with previous observationsthat lysed cells show about five-fold moreTF activity than do intact cells. These findings indicate the presence of an intracellular storage site for TF in some cell types, a pattern presently associated only with constitutive expression of this receptor protein. In addition, they confirm thatTF is induced in stimulated monocytes rather than translocation or modification. Supported by NIH grants HL-16411 and CA-41085.
THROMBOLYSIS BY TISSUE-PLASMNOCEN ACTIVATOR AND A FIBRIN (OCEN) -DEGRADATION PRODUCT, PEPTIDE 6A, IN A CANINE MDDEL OF ELECTRICALLY-INDUCED CORONARY THROMBOSIS
Authors: Saldeen T , Mehta J , Nichols W , Lew D
Published: August 2018

Intracoronary thrombus resulting in acute myocardial ischemia can be lysed by thrombolytic agents, such as, streptokinase or t-PA. We examined the potential of a recombitant tissue-plasminogen activator (rt-PA)and a fibrin (ogen)-degradation productpentapeptide 6A, Ala-Arg-Pro-Ala-Lys, corresponding to aminoacids 43-47 in the BB-chain of fibrinogen, which causes marked increase in coronary blood flow and stimulates prostacyclin release, in restoring coronary blood flow in dqgs with experimentally-induced thrombus. An occlusive thrombus was created in the circumflex (Cx) coronary artery in 8 dcgs by electricalstimulation of the endothelial surface. The electrically-induced Cx thrombus consisted primarily of platelets and fibrin. After the occlusive thrcmbus was stable without electrical currant, rt-PA (10ug/kg/minute for 30 minutes intravenously)or peptide 6A (5 unoles/minute for 20 minutes intracorcnary) were randomly administered. Infusion of t-PA restored coronar blood flow (peak 22 ±12 ml/minute, mean ±SD) in five of seven animlas. The time to flow restoration was 12.3 ± 9.1 minutes and the reflow persistedfor20.0 ± 10.9 minutes. Peptide 6A administration also restored coronary blood flow (peak 20 ± 4 ml/ minute) in seven of eight animals with occlusive coronary thrombus. Mean time to blood flow restoration (4.3 ±2.9 minutes) wasshorter(P>0.05) than with rt-PA, but thereflow persisted only for the duration of tine infusion (16.3 ± 10.2 minutes).Peptide 6A adninistration was associatedwith a significant (P±0.05) increase in plasma 6-keto-PGF1α indicating stimulation of prostacyclin release. In addition, plasma t-PA concentrations also increased (F>0.01) at the peak effect of peptide 6A indicating releaseof endogenous t-PA as another potentialmechanism of the thrombolytic effects of peptide 6A. This study demonstrates that peptide 6A exerts coronary thrombolytic effectsccmpa rable to those of t-PA in a canine model of coronary thrombosis.
RELATIONSHIP BETWEEN BLEEDING EVENTS AND CHANGES IN PLASMA IN PATIENTS TREATED WITH RECOMBINANT TISSUE PLASMINOGEN ACTIVATOR AND STREPTOKINASE IN TIMI STUDY-PHASE I
Authors: Rao MD A K
Published: August 2018

In Thrombolysis in Myocardial Infarction (TIMI) trial (Phase I) patients were randomized to receive intravenously either 80 mg of predominantly double~chain (G11021) recombinanttissue plasminogen activator(rt-PA) over 3 hrsor 1.5 million units of streptokinase (SK) over 60 min. All patients received an intravenous heparin bolus (5000 units) prior to angiography and a continuous infusion (1000 units/hr) starting 3 hr later.Bleeding occurred in 33% (rt-PA) and 31% (SK)of patients; over 70% of episodes were at vascular puncture sites. We report here the relationship between bleeding events and changes in plasma fibrinogen (FBG), plasminogen (PMG),and fibrin(ogen) degradation products (FDP) at 5 hr after initiation of drug infusion.Shown are number of patients with hemorrhage, total number of patients,and correlation coefficients (r) for occurrence of bleeding versus the changes in plasm proteins in each group.

The data indicate a relationship between bleeding events and changes in plasma proteins,with higher frequency of bleeding in patients with greater changes. FDP levels in both groups show a correlation with bleeding unlikely due to chance alone. While the hemorrhage may be due to multiple factors such as vigorous anticoagulation, arterial punctures, and platelet inhibition, the above findings suggestthat the plasma changes contribute to the hemorrhagic events.
CORONARY THROMBOLYSIS AND PREVENTION OF RE0CCLUSI0N WITH RECOMBINANT DOUBLECHAIN TISSUE PLASMINOGENACTIVATOR
Authors: Kent R
Published: August 2018

The dose response (DR) of tissue plasminogen activator (t-PA) in patients (pts) with acutemyocardial infarction (AMI) and the efficacy of 4-6 hr maintenance infusions (M) of t-PA in preventing early reocclusion (R) have been reported in small numbers of pts. No studies have determined the efficacy of longer M in limiting short term R.In two related trials weexamined DR and long term M in a large numberof pts. We administered a uniform t-PA product [>98% double chain (DC)] to 223 pts 3.1 ±1.1 hrs after the onset of AMI.Pts received 0.25 - 0.95 megaunits (MU)/kg/90min and then underwent coronary angiography(CA):Responders were defined by TIMI grades 2 or 3. Results were In a logistic regression anaylsis the coefficient for dose was significant(p<.02). Two subsets of the above pts received M. Groups A (4l pts) and B (51 pts) had received 0.26 - 0.40 and 0.4l - 0.54 MU/kg/90 min respectively. 66% of A and 78%of B were responders, received an additional 90 minof t-PA at 1/3 the initial rate and then 1 of4 M for 9-21 hrs (mean total 17.2±4.5 hrs) at which time CA was repeated. Results for A + B were:For all pts fibrinogen decreases were mild. Bleeding was primarily related to vascular invasion and M length. We conclude that the DR for DC tPA can be accurately defined and long term M to limit .short term R are feasible. Future studies may define t-PA dosing regimens suited for specific clinical situations.
RESULTS OF COAGULATION - FIBRINOLYSIS ANALYSES IN 386 PATIENTS WITH ACUTE MYOCARDIAL INFARCTION TREATED WITH RECOMBINANT TISSUE-TYPE PLASMINOGEN ACTIVATOR (rt-PA) (TAMI TRIAL)
Authors: Stump D C , Topol E J , Califf R , Chen A B , Hopkins A , Collen A D
Published: August 2018

Three hundred eighty-six patients with acute myocardial infarction received up to 150 mg rt-PA (single chain) IV either over 8 h (60mg over 1 h, 20 mg/h for 2 h,10 mg/h for 5 h)(173 pts) or over"5h(1 mg/kg over 1 h, remainder over 4 h) (213 pts),before randomization to early or late angioplasty. Blood was collected on a lyophilized mixture of citrate and the t-PA inhibitor D-Phe-Pro-Arg-CH2C1 (PPACK), to maximally prevent in vitro fibrinolytic activation and concomitant fibrinogen degradation. The plasma rt-PA level increased to 2.4±2.0 /μg/ml(mean +SD)and 1.7 ±1.3 /μg/ml after 3h and to 1.0 ±1.8 and 1.0 ±0.9 /μg/ml at the end of the infusion.Fibrinogen levels (coagulation rate assay) fell to 5 ± 28 and 52 ± 27% at 3 h and to53 ± 28 and 47 μ 26% at the endof infusion.Fibrinogen degradation productsincreased to 32 /μg/ml (median, with 10 and 90 percentile values of 2 and 512 /μg/ml) after 3h and to3 2 /μg/1 (median, with 10 and 90 percentile values of 2and 512 ug/ml) at the end of infusion. The fibrinogen decreased to below 1 g/1 in 23% of patients and b e low 0-5 g/1 in 11% after 3 h infusion with corresponding values of 33% and 12%at the end of infusion.Thus, at the infusion rates required for rapid coronary artery reperfusion in man, rt-PA remains relatively fibrin-specific. The cause of the extensive fibrinogen depletion occurring in some patients remains to be further investigated.
THE EFFECTS OF GR32191, A NEW THROMBOXANE RECEPTOR BLOCKING DRUG,ON PLATELETS AND VASCULAR SMOOTH MUSCLE IN VITRO
Authors: Lumley P , Collington E W , Hallett P , Hornby E J , Humphrey p PA , Wallis C J , Jack D , Brittain R T
Published: August 2018

The effect of a new thromboxane receptor blocking drug GR32191 ([1R-[1α(Z),2β,3β,5α]]-(+)-7-[5-[[(1,1"-biphenyl)-4-yl]methoxy] -3-hydroxy-2-(l-piperidinyl)cyclopentyl]-4-heptenoic acid,hydrochloride) has been examined upon platelets and vascular smooth muscle. In human platelet-rich plasma (PRP), aggregation to thromboxane(Tx) A2, PGH2, arachidonic acid, collagen andU-46619 was antagonised by GR32191 (IC50 range 2-36 nM).Primary aggregation (PRP treated with aspirin 10 pM) to ADP, 5-HT and adrenaline were unaffected by concentrations of GR32191 up to 10 pM. In human PRP, U-46619-induced aggregation and 5-HT release were antagonised by GR32191(10-100 nM). In contrast, in theabsence of aspirin, ADP-induced 5-HT release,but not aggregation, was antagonised by the compound implicating a role for TXA2 in the release process. In human PRP GR32191 (up to 30μM) did not itself induce aggregation or, in the presence of EGTA (4 mM), induce detectable shape change. Up to 10 μM GR32191 was without effect upon the inhibitory activity of PGI2 or PGD2 and at 1μMhad no significant inhibitory activity upon fatty acid cyclooxygenase, thromboxane synthase, prostacyclin synthase, 12-lipoxygenase orphosphodiesterase. The effect of GR32191was quantified further in human platelets suspended in whole blood or physiological salt solution. Aggregation to U-46619 was antagonised byGR32191 with a pA2 (slope of the Schild regression) of 8.2 (1.3) in whole blood and 8.8 (1.3) in resuspended platelets. The compound competitively and specifically antagonised the contractions of strips of human isolatedpulmonary blood vessels and rat and guinea-pig aortic strips produced by U-46619 with pA2 (slope) values of 8.2 (1.1), 7.9 (0.9) and 8.7(0.9) respectively. In contrast contractions induced by KC1 and 5-HT (rat) orKC1and histamine (guinea-pig) were unaffectedbyconcentrations of GR32191 up to 30 μM.Thus GR32191 is a potent and specific thromboxane receptor blocking drug on platelets and vascular smooth muscle in vitro. It is orally active and long lasting in man (Thomas, M et al.,this meeting).
THE EFFECT OF A COMBINED TXA2SYNTHETASE AND TXA2/PROSTAGLANDIN ENDOPEROXIDE RECEPTOR BLOCKER(R 68 070)ON THE ACTIVATION OF EXCITATORY RECEPTOR-COUPLEDPHOSPHOLIPASE C IN HUMAN PLATELETS
Authors: De Chaffoy de Courcells D , De Clerck F
Published: August 2018

Prostaglandin endoperoxides (PGEND) and thromboxane A2 (TxA2)contribute to the activation of platelets, involving inositol-containing phospholipids as a signal transducing system. A primary step in this signal transduction consists of the activation of phospholipase C, which then yields diacylglycerol and inositol phosphates;diacylglycerol is subsequently phosphorylated to phosphatidic acid (PA). In platelets prelabelled with [32P] orthophosphate,receptor activation is quantitatively reflected by an increased formation of [32P]-PA.Using this assay, the relativeimportance of endogenously generated PGEND and TxA2 for the subsequent PA-formation was analysed by means of pharmacological manipulations. R 68 070, an oxime-alkanecarboxylic acid derivative combining specific TxA2 synthetase inhibition with TxA2/PGEND receptor blockade in one molecule (1 x 10-6M) inhibited the formation of PA in collagen-stimulated platelets; by contrast, synthetase inhibitors without receptor effect (da-zoxiben, OKY-1581,1x 10-6M) increased the PA-response tocollagen.

Using U 46619 as a stimulus, R 68 070 as well as BM 13177, a receptor blocker withouteffect on synthetase (1 x 10-6 M)both reduced the PA-response to the same extent while OKY-1581 (1 x 10-6 M) was ineffective. The phospholipid response induced by serotonin, vasopressin or PAF were not affected by R 68 070, demonstrating its specificity for the prostanoid system.

From these data we suggest that selectiveinhibition of TxA2 synthetase does not prevent activation of excitatory platelet receptors for arachidonate metabolites. Effectiveness was only obtained by combined TxA2 synthetase/PGEND receptor blockade.
INCREASED AGGREGATION AND SECRETION RESPONSES OF HUMAN PLATELETS WHEN LOADED WITH THE CALCIUM FLUORESCENT PROBES QUIN2 AND FURA-2
Authors: Lanza F , Beretz A , Kubina M , Cazenave J-P
Published: August 2018

Incubation of human platelets with the fluorescent dye esters quin2-AM (10 μM) or fura-2-AM (1 μM) makes possible the direct measurement of intracellular free calcium ([Ca2+1).Underthese conditions, basal levels of [Ca2+]i of 120 ± 16 nM (n=23) using quin2 and 137 ± 15 nM (n=5) using fura-2 can be measured. Both probes record comparable increases of [Ca2 ]i after stimulation with ADP, thrombin, PAF, or U-46619. Incorporation into human platelets of quin2 or fura-2 at the concentrations used to monitor [Ca2+]i leads to the activation of platelets. This was shown by increased aggregation and secretion responses of quin2or fura-2 loaded platelets after stimulationwith ADP (5 μM), PAF (1 μM) and with low concentrations of thrombin (0.015U/ml), collagen (0.5 μg/ml), the endoperoxide analog U-46619 (0.5 μM) or the calcium ionophore A 23187 (1 μM). Quin2 and fura-2 mediated platelet activation could be due to altered arachidonic acid metabolism, since it was partly inhibited by prior treatment with the cyclooxygenase inhibitor acetylsalicylate (1 μM). In contrast, platelets loaded with higher concentrations of calcium chelators (20 to 100 μM quin2-AM)exhibited diminished aggregation responses to all aggregating agents. Thislatter effectwas accompanied by increased fluidity of theplatelet plasma membrane bilayer and by the exposure of a new pool of membranes at the outer surface of platelets, as monitored withtrimethylammonium-diphenylhexatriene (TMA-DPH) in platelets loaded with thenon-fluorescent calcium probe analog MAPT. Platelet shape change, as measured in the aggregometer, was dose-dependently inhibited after loading of quin2 (10-50 μM quin2-AM), even at concentrations which potentiated aggregation. We conclude that incorporation of intracellular calcium chelators alters platelet responses, including at concentrations used to monitor intracellular calcium changes.