bluuebunny/crossref_metadata_2025 huggingface dataset:
PROTEIN C TOCHIGI: AN ABNORMAL PROTEIN C CHARACTERIZED BY DEFECTIVE RELEASE OF ACTIVATION PEPTIDE BY THROMBIN
Authors: Matsuda M , Sakata Y , Sugo T , Tanabe S , Mimuro J , Murayama H
Published: August 2018

A molecular abnormality of protein C (PC) has been found in an 18-year-old female who has been thrombophilic since she was one-year old. Detailed coagulation studies revealed that PC antigen was only 25% of normal while PC activity was substantially undetectable in her plasma. Other related plasma proteins including vitamin K-dependent blood coagulation factors, antithrombinin and plasminogen were all within normal limits.

Family study demonstrated that her father, one of her paternal uncles andher half-bred sister had half-normal levels of both activity and antigen of PC. They thus appear to be heterozygotes for PC deficiency. Her mother had anormal level of PC as antigen but only60% of normal as activity. We thus presumed that nearly a half population ofPC was not properly functioning in hermother's plasma. By utilizing insolubilized monoclonal* antibodies against PC, we isolated both single-chain and two-chain forms of PC from the propositus' plasma. They were found to have normal molecular weights by SDS-PAGE, but they failed to exhibit amidolytic activity and to incorporate 3H-labeled DFP after thrombin-treatment. By immunoblotting and ELISA utilizing a monoclonal antibody that recognizes the activation peptide part of the heavy chain of PC, we found that cleavage of the activation peptide by thrombin was impaired totally in the proposi and partly in her mother’s PC. It is thus most probable that the propositus inherited PC deficiency from her father and a molecular abnormality of non-activatable protein C from her mother, respectively. Accordingly, all the PC molecules in the propositus’ plasma are non-functional, which may have culminated in the occurrence of cerebral thrombosis at the age of 1 and severe deep venous thrombosis and bilateral pulmonary embolisms at the age of 10.
INHIBITION OF HUMAN PROTEIN C ACTIVATION BY VITAMIN K-DEPENDENT PROTEINS, INVOLVEMENT OF THE γ-CARBOXIGLUTAMIC ACID DOMAIN IN DISTINCT INTERACTIONS WITH THE HUMAN THROMBIN-THROMBOMODULIN COMPLEX AND PHOSPHOLIPIDS
Authors: Freyssinet J M , Beretz A , Klein-Soyer C , Gauchy J , Gauchy J , Schuhler S , Cazenave J P
Published: August 2018

Protein C (PC) activation by thrombin (T) occurs at the endothelial cell (EC) surface in the presence of thrombomodulin (TM). Reconstitution of purified TM into phospholipid (PL) vesicles results in an increased activation of PC but not of Gla-domainless-PC (GD-PC), a chymotryptic derivative of PC lacking the γ-carboxyglutamic acid domain (Gla-domain). We show that several human vitamin K-dependent proteins can interfere in the activation of human PC by the human T-TM complex either in the presence or in the absence of PL. Prothrombin fragment 1 (F1), peptide 1 -4.1, the N-terminal chymotryptic Gla-domain of prothrombin (F II), FI I and factor X (FX) were able to inhibit PC activation. They had no effect on the amidolytic activity of activated PC. Non-competitive inhibition was observed in the presence of 10 yM F1 when PC, at various concentrations, was activated by the 8 μM T-TM complex, at 2 mM Ca2+, with or without PL-reconstituted TM. In any case the apparent Km remained unchanged at 2 μM. In the presence of optimal PL concentrations and in the absence of F1, the Vmax could be enhanced up to 9-fold. When F1 was present, the extents of inhibition with and without PL were comparable and resulted in a 3fold decrease of the Vmax. These effects were independent of Ca2+ between 1 and 5 mM and of T between 10 and 50 nM. At 0.6 μM PC, half maximal inhibition occurred at 8μM F1 and 1 μM peptide 1-41 in the presence or in the absence of PL. Protein S and factors VII and IX had only minimal effect. The inhibition due to F1 and FX was also noticed when PC was activated by T in the presence of cultured human vascular EC. A Ki of 4 μM could be determined for F1 with EC-bound TM. The non-competitive character was confirmed by the observation that F1 could also inhibit the activation of GD-PC by the T-TM complex. Incomplete heat-decarboxylation of F1 and FII, partially abolished their capacity to inhibit PC activation. These results suggest that the Gla domain of PC is involved in two distinct types of interactions. This vitamin K-dependent functional entity is necessary to allow the enhancement of PC activation by anionic PL and also interacts with the T-TM complex.
MONOCLONAL ANTIBODIES OF THE IgM AND IgG CLASS SPECIFIC FOR CROSSLINKED FIBRIN DEGRADATION PRODUCTS
Authors: Gaffney P J , Creighton L J , Curry A , MacMahon B , Thorpe R
Published: August 2018

Monoclonal antibodies (mabs) to crosslinked fibrin degradation products (XL-FDP) having the general formula D/Y[X]nY/D (known as X-oligomer) and D-D (known as D dimer) have been raised in balb/C mice by both a novel mtrasplenic and a conventional subcutaneous route of immunisation and by combinations of both these procedures. Mabs to X-oligomers (NIBn 52 and NIBn 123) obtained by an intrasplenic procedure have been demonstrated to crossreact only with X-oligomer in a 2-site ELISA procedure and not with D dimer or whole fibrinogen and have been shown to be of value m the examination of clinical material obtained from patients with various types of thrombosis and have also been useful in monitoring the efficacy of thrombolytic therapy. The X-oligomer mabs are immunoglobulins of the M class. It was demonstrated that their unique specificity for conformational epitopes on the large X-oligomer fragments does not reside in the IgM structure since alterative immunisation procedures have been used to generate mabs of the IgG class which have the same specificity. Using immunoglobulin class switching in culture rather than during immunisation was suggested by certain cell lines which produced both IgM and IgG specific for X-oligomer. This latter point needs rigorous validation.

Immunoglobulin G type mabs to highly purified D dimer were raised by conventional subcutaneous immunisation of balb/C mice. One of these, NIBn-11, was found to crossreact with PVC-immobilised X-oligomer and D dimer but not with fibrinogen. However NIBn-11 did not bind to D dimer in a 2-site ELISA procedure while crossreactmg quite avidly with X-oligomer. This suggests that the D dimer epitope to which NIBn-11 is directed is expressed in some conformations and not m others and that these conformations are always expressed in the complex X-oligomer group of fragments. These mabs, whilst of value in measuring certain unique fibrin fragments m plasma, are useful in the epitope mapping of fibrinogen/fibrin and their plasmm-mediated
ENZYMATIC AND IMMUNOLOGIC CHARACTERIZATION OF A CYSTEINE PROTEINASE PROCOAGULANT IN SEVERAL MURINE METASTASIZING TUMO
Authors: Roncaglioni M C , Falanga A , Bolognese Dalessandro A P , Casali B , Donti M B
Published: August 2018

Involvement of the hemostatic systemin tumor metastasis growth has been repeatedly suggested and several tumor-associated procoagulants have been described. We have studied here the procoagulant activity (PCA) of tissue extracts from 4 murine metastasizing tumors: Lewis Lung Carcinoma (3LL), B16 melanoma (B16), JW sarcoma (JWS) and the M4 variant of the mFS6 fibrosarcoma (M4). The experiments were designed to identify cancer procoagulant (CP) a FVII independent FX activating cysteine proteinase or tissue factor (TF) in these tumors. Tissue extracts from 3LL, B16 and JWS initiated coagulation both in the presence and absence of FVII (FVII independent activity ranging from 70% to 86% of the total activity). The PCA of the same tumors was significantly decreased (p < 0.01) by cysteine proteinase inhibitors (1 mM iodoacetamide (IA) and 0.1 mM HgCl2 ) and the inhibitionby HgCl2 was reversed by -SH group activators (di-thiatreital, KCN, IiDTA). In addition these samples were able of directly activating pure bovineFX in a two stage clotting assay. The PCA of M4 extract was dependent on FVII,was not significantly affected by IA and HgCl2 and was inhibited by concanavalin A, a known TF inhibitor. An Ouchterlony double immunodiffusion study showed immunological cross-reactivity of 3LL, B16 and JWS to a polyclonal antibody to purified CP (from rabbit V2 carcinoma; obtained from S.G. Gordon, Denver, USA). No cross-reactivity was present between this antibody and M4. This study shows that the PCA of M4 is TF, whereas the procoagulant(s) of 3LL, B16 and JWS are enzymatically and immunologically indistinguishable from CP.
PURIFICATION AND CHARACTERIZATION OF TWO PROCOAGULANTS FROM WALKER 256 CARCINOSARCOMA TUMORS,
Authors: Gordon Stuart , Sloane Bonnie , Cavanugh Phil , Cross Barbara , Honn Kenneth , Chelladurai Mohanathasan
Published: August 2018

Activation of the coagulation system bytumor cells may play an important role in tumor growth and metastases. Becauseprocoagulant activities have been identified in different tumor cells by different investigators, effective comparison of these activities has been difficult. Therefore, we purified and characterized two different procoagulant proteins from the same Walker 256 tumors. The first procoagulant activity/platelet aggregating activity (PCA/PAA) was purified from a 1% CHAPS detergent extract oftumor homogenate followed by (NH4)2SO4 fractionation, anion exchange and hydrophobic chromatography. The protein had a molecular weight of 58,000, required phospholipid and an intact coagulation pathway from factor X through fibrinogen for activity, but did not require factors VII or IX forits procoagulant activity. The procoagulant activity was not inhibited by 5mMphenyl-methyl sulfonyl fluoride, iodoacetamide or phenanthroline; there was noevidence of proteinase activity. The PAA was due to thrombin generation during coagulation. The second procoagulant,cancer procoagulant (CP), was extracted from tumors in barbital buffer (pH 7.4) without detergent, purified by immunoaffinity (using a polyclonal goat antibody to CP from V2 carcinoma) and mercurial-benzoate affinity chromatography. CP had a molecular weight of 68,000, an isoelectric point of 4.8 and initiated coagulation by directly activating factor X in the coagulation system. CP was inhibited by Hg++ and iodoacetamide, cysteine proteinase inhibitors. The purified CP formed an immunodiffusion precipitin band against the polyclonal anti-CP goat antibody. Thus, thepurified CP had the same physicochemical, enzymatic and immunologic propertiesas CP from rabbit V2 carcinoma. Neither procoagulant had the properties of tissue factor. These results suggest that there aretwo distinct procoagulant activities inWalker 256 and that both may contributeto the coagulation abnormalities that are associated with tumor growthand metastases.
EXTRAVASAL FIBRIN STABILIZATION BY FACTOR XIII IN LYMPH NODES WITH HODGKIN’S DISEASE
Authors: Adày R , Szegedi A , Nemes Z , Muszbek L
Published: August 2018

The formation of extravasal fibrin deposits in various tumors has been recognized a long time ago and it has been implicated in various aspects of tumor growth. However, no adequate information is available on the nature of intratumoral fibrin. In this study we attempted to find out if fibrin deposit in human lymph nodes with Hodgkin’s disease is stabilized and made resistant to fibrinolysis by factor XIII /FXIII/ of blood coagulation. The two main tasks for FXIII in fibrin stabilization is to attach a^antiplas-min the main phyiological inhibitor of fibrinolysis to fibrin strands and crosslink fibrin chains. By double immunofluorescent labeling for fibrin and α2-antiplasmin a complete colocalization of the two antigens could be observed. A part of fibrin strands also stained for α2-antiplasmin-plasmin-complex-neoantigen revealing that α2-antiplasmin covalently linked to fibrin inhibited intratumoral fibrinolysis. The finding that immunolabeling for fibrin was preserved following the treatment of sections by concentrated urea solution clearly demonstrates that fibrin chains became crosslinked by FXIII. These results were further supported by SDS PAGE analysis of intratumoral fibrin deposits. There are two theoretical possibilities for the appearance of FXIII in the interstitial space: 1/ plasmatic FXIII can get acrossthe vessel wall when increased permeability is induced 2/ FXIII can be produced and released by certain cells of the tumorous tissue. We explored the secondpossibility by various immunomorphological and enzymcytochemical techniques. Alarge number of FXIII positive cells were detected by immunoperoxidase technique in the follicular and interfollicular region of malignant, but not in normal lymph nodes. These relatively large, multipolar, mononuclear cells possesseda macrophage-like appearance and showedANAE-positivity,i.e.,they belong to thegroup of tumor associated macrophages. FXIII containing cells were labeled by monoclonal anti-Leu M3 (a monocyte/macrophage marker), but not by anti-HLA-DR.They were often found in the immediate vicinity of malignant Hodgkin’s cells and also showed an intimate relationshipwith extravasal fibrin formation.lt is suggested that FXIII secreted byintactor released from damaged macrophages might be involved in the stabilization offibrin in the tumor stroma or around tumor cells.
FIBRINOGEN INFLUX AND FIBRIN ACCUMULATION IN ASCITES TUMORS
Authors: Nagy J A , Devorak H F
Published: August 2018

Fibrin gel matrix organizes solid tumors into discrete cell nests and providesa provisional matrix for mature stroma generation. Fibrin deposits result fromlocal extravasation of plasma fibrinogen followed by extravascular coagulationand crosslinking. Unlike solid tumors, ascites tumor cells in body cavities grow in suspension, and are not envelopedin fibrin gel. The lack of fibrin gel in ascites tumors has several possible explanations: 1. Peritoneal wall blood vessels are impermeable to molecules as large as fibrinogen. 2. Fibrinogen leaks from peritoneal vessels, but is clotted to fibrin and never reaches the peritoneal cavity. 3. Fibrinogen leaks from vessels, reaches the peritoneal cavity, and is rapidly degraded. To distinguish among these possibilities we investigated the permselective properties of the peritoneal wall. We measured the influx of intravenously (iv) administered, fluorescein-labeled dextrans (FITC-D) of varying size into the peritonea of normal and tumor or inflammatory ascites-bearing mice and guinea pigs. FITC-D of MW 70-5,000 kD leaked from vessels and ∽ 10-fold more entered MOT or TA3/St tumor-associated or serotonin-induced inflammatory ascites than normal peritonea. In tumor bearing animals, 3-20% of injected FITC-D entered the peritoneum within 15-30 min., all of it intact by gel exclusion chromatography. We also investigated the influx of fluorescein-labeled fibrinogen (FITC-F). At 30-60 min after iv injection, FITC-F hadextravasated into the peritoneal wall as judged by fluorescence microscopy (tumor bearing ≫ normal animals). Moreover, monoclonal antibodies reactivewith fibrin, but not fibrinogen, stained such deposits by immunohistochemistry in guinea pigs bearing line 1 or line 10 ascites tumors. Following iv injection of 125l-fibrinogen, as much as 11% of injected radioactivity accumulated in tumor ascites fluid over1-3 hrs (versus ∽1% in normal controls). In both, only ∽half was clottableand 3040% was of low MW (≤.10 kD). In contrast, iv 125I-albumin also entered the peritoneal cavity at an enhanced rate but showed no evidence of degradation. We conclude that fibrinogen extravasates from peritoneal vessels,thatleakage is increased in ascites tumor-bearing animals, and that fibrinogen clots, at least in part, before it enters the peritoneal cavity. Also, fibrinogen undergoes selective degradation and much of the fibrinogen that does enter the peritoneum represents degradation products of fibrinogen/fibrin. The resulting lack of peritoneal fibrin gel matrixfavors the suspension pattern of ascites, as compared with solid tumor growth.
THROMBIN-ANTITHROMBIN III (TAT) COMPLEXES: A STUDY IN CONSECUTIVE PATIENTS SUSPECTED OF DIC
Authors: Hoek J A , Lamping R , Sturk A , Borm J J J , Berends F , tenCate J W
Published: August 2018

DIC is a clinical syndrome which is initiated by exces sive thrombin generation, fibrin formation and fibrin deposition in organs, thus resulting in multiple organ failure. It is a frequent, often fatal complication in intensive care patients. We evaluated a recently developed ELISA (Behringwerke) for TAT complexes in 50 healthy volunteers and in 43 consecutive intensive care patients referred by the attending physician on the clinical suspicion of DIC. The ELISA is based upon thrombin-antibody coated test tubes and measurement of bound TAT complexes with peroxidase-conjugated AT III antibody. The method has a detection limit of 0.7 μg TAT/L, a normal range of 0.97-5.4 μg/L, The intra- and inter assay v.c. at 3.3, 11.2 and 42.4 μg/L TAT levels were 5, 7, 8% and 9 , 9 and 12% resp. Patients suspected of DIC were in a clinically septic condition (n=33), confirmed by positive bloodcultures in 18, malignancies (n=5), multiple trauma (n=3) or solutio placentae (n=2). Of the 43 patients 29 had increased TAT levels: mean 21.5, range 5.4-62.5. TAT levels correlated (p <0.05) with concurrently assayed plasma fibrinogen (r=0,543), Factor V (r=0,539), platelet count (r=0,307), AT III (r=0,317), and with plasminogen (r=0,365) (Spearman rank correlation test).
The accuracy of the TAT-test for the diagnosis of DIC was assessed in all patients. DIC was assumed if patients fulfilled 3 of the 5 following laboratory criteria, i.e. AT III <0.7 U/ml, factor V <0.6 U/ml, fibrinogen <1.5 g/L, platelet count <100x109/L or a positive fdp/FDP test. The sensitivity and specificity of the TAT-test was 86 and 46% respectively, the positive and negative predictive values were 60 and 77%.
ORGANON 10172 VS. WARFARINTO PREVENT VENOUS THROMBOSIS AFTER HIP FRACTURE
Authors: Gerhart T , Yett H , Donovan A , Lee M A , Smith M , Salzman E W
Published: August 2018

Deep venous thrombosis (DVT) remains a serious and frequent complication after fracture of the hip, and even the mostefficacious prophylactic agents, e.g., warfarin, may fail to prevent DVT in up to 2056 of cases. There is evidence that low molecular weight heparin or heparin-like agents may have advantages in antithrombotic prophylaxis with reduced hemorrhagic sideeffects in patients at risk of DVT. We are engaged in a randomized prospective trial comparing the antithrombotic effect ofwarfarin (PT 1.5x control)with that of Organon 10172, a mixture ofsulfated low molecular weight glycosamioglycans (750 anti-Xa u b.i.d. sc., begunpreop and continued 9 days, followe by warfarin). Diagnosis is by 125-1 fibrinogen scan and impedence plethysmography with confirmatory phlebography. At present71 patients have been admitted, and patient groups are comparable in age, sex, type of fracture, and all other significant respects. DVT has been diagnosed in 7of 36 patients given warfarin an in 1 of35 patients who received Organon10172. Pulmonary embolism has not beenencountered. GI bleeding has occurred twice on warfarin and once on Organon 10172 There has been no difference in estimated operative blood loss, transfusion requirements, or other major bleeding complications.One patient on warfarin died ofmyocardial infarction and pneumonia. There were no other adverse reactions.

The study is still in progress. The present trend in the results suggests that the heparinoid Organon 10172 may be a promising new agent to prevent DVT in high risk patients, such as those with fractures of the hip.
DIHYDROERGOTAMINE-HEPARINPROPHYLAXIS OF DVT IN TOTAL HIP REPLACEMENT PATIENTS: A MULTICENTER TRIAL
Authors: Sasahara A A , Diserio F J
Published: August 2018

We report on the results of a controlled, blinded, U.S. multicenter trial in theprophylaxis of postoperative DVT in total hip replacement patients. One hundred and forty-eight (148) patients were enrolled in this evaluation of two parallel treatment groups: (1) dihydroergotamine 0.5 mg with heparin 5000 units(DHE/5000),or (2) matching placebo. Treatment was administered subcutaneously preoperatively and continued three times a day (Q8h)for at least seven days. One hundred andtwenty-eight (128) of these were valid protocol compilers.

Daily radiofibrinogen leg scans performed for the duration of treatment were followed by venographic evaluation on postoperative Day 7 or when the RFUT indicated a sustained positive reading for 48 hours. In 12 patients venography was not performed and these patients are not included in the efficacy analysis, as were eight other protocol violators. The venographically confirmed positive DVT rates for the consultant's review (protocol compilers) were 25% (16/63) for DHE/5000 and 52% (32/65) for placebo, a statistically significant difference in effective prevention of DVT (p=0.0021, two tailed Fisher's exact test)The incidence of proximal thrombi (popliteal and above) was reduced from 18.5% (12/65 patients) in the placebo group to 4.8% (3/63patients) in the group treated with DHE/Heparin (p=0.0255), while the incidence of extensive thrombi (<25% occlusion) was reduced from 24.6% (16/65 patients) to9.5% (6/63 patients) in the respective groups (p=0.0399).

The most common side effect was injection site hematoma occurring in seven of the DHE/5000 patients and two of the placebo patients. One DHE/5000 patient discontinued treatment due to a side effect (injection site hematoma) attributable to drug and three placebo patients discontinued treatment due to side effects (two of these had confirmed pulmonary embolism).

These data confirm previous investigations supporting the efficacy and safetyof DHE/5000 for the prevention of postoperative DVT in patients undergoing totalhip replacement.
THE ISRAELI STUDY OF EARLY INTERVENTION IN MYOCARDIAL INFARCTION; INTRAVENOUS RT-PA WITH SUBSEQUENT REVASCULARIZATION.
Authors: Barbash G I , Hod H , Miller H I , Roth A , Rath S , Har Zahav Y , Rabinovitz B , Laniado S , Seligsohn U
Published: August 2018

Immediate mechanical revascularization forlarge population of acute myocardial infarction (AMI) patients, is logistically impractical. Effectiveness of early intravenous rt-PA,and feasibility of delaying the coronary angioplasty were studied. 57 AMI patients were enrolled since October 1986; 30 via Emergency ward, and 27 via mobile intensive care unit. The mean time interval from onset of ischemic pain to rt-PA bolus was 115+52 min.The protocol included an initial 10 mg rt-PA bolus followed by continuous infusion of 110 mg over 6 hr, concomitant continuous heparin and lidocain infusion, and aspirin 250 mg/day; coronary catheterization after 72 hr. and angioplasty of suitable infarct-related artery (IRA). 49 patients (86%) had clinical signs of reperfusion (disappearance of chest pain with resolution of ST elevation) within 60 min. The 8 "non-responders" were treated earlier (shorter time interval from onset of painto rt-PA bolus) than the "responders"(87+37 and 120+53 min respectively,p=0.01). Of "responding" patients, 5 had intermittant reocclusion-reperfu-sion cycles, and an additional 4 reoccluded silently before coronary catheterization (7 to 72 hr).29 PTCA successful procedures(53% of pats) of the infarct-related artery (IRA) were performed: 25 during the protocol catheterizationat 72 hr, and 4 which were performed as an emergency procedure of high grade stenosis (1),or totally occluded IRA (3), in 4 of the 8 "non-responders".There were 3 emergency and 8 elective bypass operations (20%). While in the 25 patients with anterior wallinfarction the admission Lt. ventricular ejection fraction (LVEF) (37+12%) improved (46+17%) at discharge, in the 30 patients with inferior wall infarction the admission and discharge LVEF were both normal.Two patients expired; one within minutes after treatment initiation, and the other following no response to the thrombolytic therapy and reocclusions of repeat coronary dilatations. These results indicate that rt-PA thrombolysis is a safe treatment modality enabling planning of deferred mechanical revascularization under more optimal conditions.
CRYSTALLOGRAPHIC INVESTIGATION OF ANTITHROMBIN III.
Authors: Samama J P , Delarue M , Moras D , Petitou M , Lormeau J C , Choay J
Published: August 2018

The plasma protein inhibitor anti thrombin III in its native form has been crystallized using standard techniques.The crystals diffract to about and belong to space group P41212 with cell parameters:a = b = 90.6<, c = 380.7<.The asymmetric unit contains three molecules of anti thrombin III.

The self rotation function computed with the native data set indicates the presence of a non crystallographic three fold axis. Cross rotation function calculations using themodel of the cleaved α1,-antitrypsin (H. Loebermann at.,J. Mol. Biol.(1985) 177, 531) suggests tertiary structuresimilarities between the two plasma proteins.This is in agreement with the already described primary sequence homology of these glycoproteins but at variance with the model of active α1-anti trypsin inferred from the previous studies on the cleaved molecule.

The technical assistance of M. Maman is deeply appreciated.
INCREASED PROTEIN C AND FIBRINOPEPTIDE A CONCENTRATION IN PATIENTS WITH ANGINA
Authors: Gensini G F , Rostango C , Abbate R , Favilla S , Mannucci P M , Neri G G , Sernery S
Published: August 2018

The present study has been designed to investigate protein C (as protein C antigen) and fibrinopeptide A (FpA) concentration in plasma from patients suffering from ischemic heart disease in relation to the frequency of ischemic attacks , in order to estabilish if modifications in protein C levels could contribute to blood clotting activation. Protein C and FpA levels in plasma were measured in 30 controls and in two groups of patients with angina. The first group was formed by 27 patients suffering from spontaneous ischemic attacks (active angina). The second one was formed by patients who previuosly suffered from angina, but were free from myocardial ischemic attacks for at least one month (inactive angina). Protein C (measured by electroimmunoassay) was higher in the whole group of patients than in controls (122.1+ 20.2 vs. 96.5+±14 p<0.001)? Moreover significantly higher values were found in patients with active angina in comparison to patients with inactive disease (132.5+±15.7 vs 112.3+±17.6,p<0.001).Similarly patients suffering from active angina had FpA levels higher than patients with inactive angina (8.6+±9.4 vs 5.5+±7.0,p<0.01) or controls (1.6+±1.0,p<0.001). A high concordance (76%) between protein C and FpA levels exceeding normal limits was found in patients with active angina (p < 0.05) but no statistically significant correlation existed between protein C and FpA levels and between protein C or FpA levels and coronary pathoanatomy. These results confirm a significant involvement of blood clotting system in ischemic heart disease and especially in active angina.
INFLUENCE OF DIETARY FISH ON PLATELET FUNCTION AND THROMBOXANE FORMATION
Authors: Houwelingen A C V , Hennissen A , Verbeek-Schlppers F , Simonson T , Fischer S , Hornstra G
Published: August 2018

Many studies have been performed with respect to the effects of fish (products) on the possible prevention of ischemic cardiovascular disease in man. Most of the trials, however, were poorly designed without a proper control group, and their results are equivocal. We, therefore, performed a well- controlled intervention trial to investigate the effect of a reasonable amount of dietary fish on certain risk indicators of arterial thrombogenesis. In Tromstf, Maastricht and Zeist, healthy male volunteers were given a dietary supplement consisting of 135 g of canned mackerel (n=40) or meat paste (control, n=42) per day for a period of 6 weeks. Compliance was monitored on the basis of the urinary excretion of lithium, added to the supplements. Average compliance was about80% and decreased slightly with time. Bleeding time was significantly prolonged and platelet number decreased in the mackerel group.Platelet aggregation inPRP induced by thrombin decreased only at a low dose.Collagen-induced platelet aggregation in PRP decreased significantly. This was associated with a 50% reduction of the collagen-induced TxB2 formation in PRP(P < 0.001). TxB3 synthesis increased significantly in the mackerel group from 0.9 to 7.8% of the TxB2 production (GC/MS). Collagen- induced platelet aggregation and ATP release in whole blood were measured with the Chronolog whole blood lumiaggregometer (Maastricht only). In the same samples TxB2 formtion was measured (RIA) and although a significant reduction was seen in the mackerel group, platelet functionswere not significantly altered. However, platelet release (but not aggregation) was significantly related to the compliance and a decreasing effect of the mackerel supplement was observed in those 50% of the volunteers having the highest compliance. This demonstrates the necessity of monitoring compliance by objective means. Acknowledgements: Financial support was obtained from the Dutch Heart Foundation. The International Association of Fish Meal Manufactures (Potters Bar, Herts, England) provided the mackerel.
rt-PA THROMBOLYSIS VERSUS SURGICAL THROMBECTOMY OF PERIPHERAL BYPASS GRAFTS:A COMPARATIVE TRIAL
Authors: Graor R , Young J , Beven B , Hertzer N , Krajewski L , O'Hara P , Olin J , Ruschhaupt W
Published: August 2018

Twenty-nine of 33 patients (88%) with thrombosed lower extremity bypass grafts had angiographic and clinical successful lysis of graft thrombi with rt-PA. Of the successful group, 18 were saphenous vein grafts and 11 PTFE grafts in the femoropopliteal-tibial position. Following lysis, 76% required a secondary procedure (2 PTA, 20 surgical repair and 7 required anticoagulation) to maintain patency.
A matched cohort of patients with bypass grafts who had surgical thrombectomy were compared to the rt-PA successfully treated grafts and analyzed for duration of patency after opening and limb salvage. The Kaplan-Meier curve compared both treatment groups and demonstrated improved graft survival in the rt-PA treated group (p=.01) (median graft survival rt-PA 195 days, surgery 30 days). Limb salvage was marginally significant (p=.064) in favor of the rt-PA treatment group. Single and multi-variant risk factor analysis found smoking and age of the graft adversely affected patency (p=.05 and p=.08 respectively). Graft type, age of the patient, diabetes mellitus, and high blood pressure were not significant factors (p> .15).

Systemic fibrinolysis was identified to varying degrees. Mean decreases in the fibrinolytic constituents include: 59% decrease in clottable fibrinogen, 18% decrease in sulfite fibrinogen, 78% decrease in alpha-2 antiplasmin and varying degrees of increases in DDIMER, B-Beta1-42 and B-Beta15-42 coincident with the constituent changes.
Complications were unrelated to constituent changes. One patient had major bleeding secondary to graft anastomosis disruption.

Thrombolysis with rt-PA is an effective and more durable adjunct treatment option for thrombosed bypass grafts, especially when combined with PTA or surgical repairto maintain patency.
FIBRINOLYTIC THERAPY IN ACUTE VERTEBROBASILAR STROKE
Authors: Del Zoppo G J , Bruckmann H , Ferbert A , Drummen R , Hacke W , Hacke W , Zeumer H
Published: August 2018

Local intra-arterial administration of fibrinolytic agents has been successfully used to achieve recanalization in acute thrombotic stroke patients (Zeumer, H., J Neurol 231:287-294, 1985).65 consecutive patients with clinical signs of severe brainstem ischemia and angiographically demonstrated vertebrobasilar (VB) thrombotic occlusion were treated with antithrombotic therapy.

22 patients (Group A) received antiplatelet/anticoagulant treatment. 43 patients (Group B) received local intra-arterial infusion of streptokinase or urokinase proximal to the thrombotic occlusion. In 19 patients of Group B (Group B1) arterial recanalization was achieved as demonstrated angiographically; in 24 patients (Group B2) the arterial occlusion could not be resolved. None of the patients in Group B2 survived.

When clinically favorable (minimal/moderate deficit) and unfavorable (severe deficit/demise) outcomes are compared, the results are highly significant (B1 vs A; p <0.007; B1 vs B2; p <0.0003 .

It was possible to describe the vascular conditions associated with angiographically unsuccessful fibrinolytic therapy (Group B2) and to identify the clinical conditions associated with an unfavorable clinical outcome in patients with successful lysis (Group B1). These data indicate that successful fibrinolytic therapy is associated with a beneficial clinical effect in VB thrombotic stroke.
TREATMENT OF ACUTE MASSIVE PULMONARY EMBOLISM WITH INTRAVENOUS VS. INTRAPULMONARY ARTERY ADMINISTERED RECOMBINANT TISSUE-TYPE PLASMINOGEN ACTIVATOR
Authors: Verstraete M
Published: August 2018

Recombinant tissue-type plasminogen activator (rt-PA) was given to 34 patients with acute massive pulmonary embolism of less than five days and with an angiographic Miller index greater than 15. The regimen was 50 mg rt-PA over 2 hours followed by repeat angiography, and, if the Miller index was judged still to be above 15, by an additional dose of 50 mg over 5 hours. Heparin was given in a bolus of 5000 IU followed by 1000 IU per hour. The rt-PA preparation (Boehringer Ingelheim GmbH, G 11021) contained mainly two-chain rt-PA. The infusion route was at random in a peripheral vein (IV) or in the pulmonary artery (PA). Pulmonary angiographs were assessed blindly by a panel of five radiologists according to the protocol developed by Miller (Br Med J 1971). 19 patients were given rt-PA via the pulmonary artery, 15 patients received rt-PA intravenously. Eleven patients were postoperative (PA 9, mean 13± ]] SD days, IV 4, 7±3 days

Sixteen patients (66%) received two infusions (PA 14; IV 8). The Miller index decreased from 25 ± 3 to 22 ± 6 (−12%) in the PA group and from 26 ± 2 to 22 ± 5 (−15%) in the IV group after the first infusion. In the patients who received a second infusion, the Miller index decreased to 16 ± 6 (−38%) after 100 mg in the PA group and to 16±;6 (−38%) in the IV group. The mean pulmonary artery pressure decreased from 30 ± 7 to 22 ± 6 (−27%) and further to 14 ± 5 (−53%) in the PA group and from 29 ± 7 to 22 ± 9 (−24%) after 50 mg and to 13 ± 5 (−55%) after 100 mg in the IV group. All these differences are significant (p < 0.01)

Fibrinogen levels dropped 46% from baseline after 50 mg and 66% from baseline after 100 mg. Bleeding occurred in 16 patients, 5 of whom had recent surgery (mean 8 days, range 2-13)

This pilot trial indicates that a prolonged infusion of rt-PA over 7 hours (100 mg) is superior to a single infusion over 2 hours (50 mg) and that infusion in the pulmonary artery does not offer a significant clinical benefit over the intravenous route. In case of life-threatening massive pulmonary embolism, treatment with rt-PA could be envisaged as early as from the third day after major surgery
CLEAVAGE PATHWAY,AND SPECIFICITY OF LEUCOCYTE ELASTASE AS COMPARED TO PLASMIN DURING FIBRINOGEN DEGRADATION
Authors: Goretzki L , Miller E , Henschen A
Published: August 2018

Plasmin and leucocyte elastase are regarded as the two medically most important fibrin(ogen)-degrading proteolytic enzymes. There is, however, a considerable difference in information available about the cleavage specificities and fragmentation pathways of these two enzymes. Degradation by plasmin has been studied already for a long time in great detail so that now the time course of the degradation, the cleavage sites and the functional properties of many fragments are well known. In contrast, relatively little is known about the degradation by leucocyte elastase, except that the overall cleavage pattern resembles that obtained with plasmin

In this investigation the leucocyte elastase-mediated degradation of fibrinogen has been examined by means of proteinchemi-cal methods. Human fibrinogen was incubated with human enzyme material for various periods of time and at some different enzyme concentrations. The split products formed at the various stages were isolated in pure form by gel filtration followed by reversed-phase high-performance liquid chromatography. The fragments were identified by N-terminal amino acid sequence and amino acid composition. The course of the degradation was also monitored by sodium dodecylsulfate-polyacrylamide gel electrophoresis. All cleavage patterns were compared with the corresponding patterns from plasmic degradation. It could be confirmed that X-, D- and E-like fragments are formed also with elastase. However, several early elastolytic Aα-chain fragments are characteristically different from plasmic fragments. The previously identified N-terminal cleavage site in the Aα-chain, i.e. after position 21, was found to be the most important site in this region of fibrinogen. The very early degradation of the Aα-chain N-terminus by elastase is in strong contrast to the stability against plasmin. Several cleavage sites in N-terminal region of the Bβ-chain were observed, though the low amino acid specificity of elastase partly hampered the identification. The γ-chain N-terminus was found to be as highly stable towards elastase as towards plasmin. The results are expected to contribute to the understanding of the role of leucocyte elastase in pathophysiologic fibrino(geno)lysis
THE WISTAR-FURTH RAT: AN ANIMAL MODEL OF HEREDITARY MACRO THROMBOCYTOPENIA
Authors: Jackson C W , Hutson N K , Steward S A , Edwards H H
Published: August 2018

The mechanisms which determine and regulate platelet size are unknown. By phase microscopy, we serendipitously observed that Wistar/Furth (W/F) rats had macrothrombocytopenia. In this study, we have characterized and compared platelets and megakaryocytes (MK) of W/F rats to those of Wistar (W), Long-Evans hooded (LE) and Sprague-Dawley (SD) rats. In addition, we have examined the mode of inheritance of this W/F rat platelet abnormality. Average platelet count of W/F rats was only 312 ± 57 x 103/mm3 compared to 1086 ± 68, 868 ± 91 and 926 ± 82 x 103/mm3 respectively for W, LE and SD rats. Mean platelet volume (MPV) of W/F rats (8.1 ± 1.4 fl) was twice that of W (4.0 ± 0.2 fl), LE (4.2 ± 0.3 f1) and SD (4.1 ± 0.2 f1) rats. However, average MK diameter and DNA content distribution of W/F rats were not significantly different than those of LE rats. Average MK concentration was somewhat lower (4.5 MK/high power field) in the W/F strain compared to that of LE rats (6.6 MK/HPF). Maze-like membrane formations were observed in W/F MK and platelets by electron microscopy. Reciprocal crosses of W/F and LE rats resulted in offspring with MPVs (4.2 fl) and platelet counts like those of LE rats, indicating that the macrothrombocytopenic trait is recessive in its inheritance. Reciprocal marrow transplants between the W/F and LE strains resulted in MPVs like those of the donor strain indicating that the macrothrombocytopenia is an intrinsic marrow abnormality of the W/F strain. Analysis of W/F platelets by two-dimensional polyacrylamide gel electrophoresis revealed two readily detectable differences from those of W and LE rats: (1) absence of a protein of MW of 41,000 and pi of 6.35 and (2) presence of a protein of MW of 35,000 and pi of 5.9. In summary, the W/F rat has a hereditary macrothrombocytopenia which is recessive in nature and not due to differences in MK size or DNA content. These results suggest that the macrothrombocytopenia of W/F rats results from formation of fewer platelets per MK, possibly due to a qualitative or quantitative defect in some component necessary for proper subdivision of megakaryocyte cytoplasm into platelets.
COVALENT Mr ∼92,000 HYBRID PLASMINOGEN ACTIVATOR DERIVED FROM THE PLASMIN FIBRIN-BINDING DOMAIN AND THE TISSUE PLASMINOGEN ACTIVATOR CATALYTIC DOMAIN
Authors: Robbins K C , Boreisha I
Published: August 2018

A covalent hybrid plasminogen activator was prepared from the sulfhydryl forms of the NH2-terminal A chain of human plasmin (Pln^) containing the fibrin-binding domain, and the COOH-terminal B chain of tissue plasminogen activator (t-PAB) containing the catalytic domain. The PlnA (SH)2 and t-PAB(SH) chains were mixed in a 1:1 molar ratio, and hybridization was allowed to proceed at 4 °C for 6 days. The covalent PlnA-t-PAB hybrid activator was isolated from the mixture by a two-step affinity chromatography method, with L-lysine-substituted Sepharose and Zn-chelated agarose. The protein yield of purified hybrid was 10% with a major component (77%) of Mr ∼92,000. The covalent PlnA-t-PAB hybrid activator, contained 1 mol of each chain; after reduction, it gave the two parent chains, PlnA and t-PAA, also shown to be present by double immunodiffusion. The specific plasminogen activator activity, with soluble fibrin, and the specific amidolytic activity, of the purified covalent hybrid activator was determined to be 200,000 IU/mg of protein, about 40% of the specific activity of the parent t-PA. In a fibrin clot lysis assay, the covalent hybrid activator and t-PA have similar specific fibrinolytic activities, 500,000 IU/mg of protein; however, the clot lysis time curves were not parallel. The binding of the covalent PlnA-t-PAB hybrid activator and t-PA to forming fibrin were found to be similar; at physiological fibrinogen concentrations, binding of both activators to forming fibrin was about 90%.
MUTANT AND CHIMAERIC RECOMBINANT PLASMINOGEN ACTIVATORS
Authors: Piérard L , Jacobs P , Gheysen D , Hoylaerts M , Cravador A , Herzog A , Collen D , Bollen A
Published: August 2018

In order to produce plasminogen activators (PA) more specific and more active than their natural counterparts, we designed recombinant genes encoding mutant forms of urokinase (u-PA) and chimaeric molecules combining fragments of tissue type plasminogen activator (t-PA) and of u-PA. The following constructs have been realized : 1°) u-PA where amino acids Arg156 and Lys158 have been replaced by Thr. The purpose of this approach was to obtain a prourokinase molecule displaying similar properties as the natural single chain urokinase (scu-PA) but resistant to the cleavage by plasmin ; 2°) u-PA where the second cleavage site, Lys135-Lys136, was also eliminated either by replacing amino acid 132 to amino acid 147 by a shorter link (Ser-Thr) as found in t-PA, or by replacing the two lysines by glutamine residues. The resulting molecules correspond thus to completely uncleavable scu-PA forms ; 3°) an hybrid composed of the finger domain of t-PA and of the B-chain of u-PA ; 4°) an hybrid made of the A-chain of t-PA and of the B-chain of u-PA ; 5°) an hybrid where the kringle 2 of t-PA has been inserted between the kringle domain and the B-chain of u-PA. The last three constructs have been made to confer the fibrin binding specificity of t-PA to the B-chain of u-PA.

All recombinant DNAs were introduced, via an expression vector, into R1610 and CosI cells. Secretion of the recombinant products was monitored by ELISA and activities were assayed in an immobilized system involving a monoclonal antibody (AAU2) raised against 33K u-PA, plasminogen and the specific chromogenic substrate S2251. In this assay, all recombinant products, except the plasmin resistant (156-158) scu-PA, showed apparent specific activities comparable to the activity of natural two-chain u-PA. Potential interest of these new plasminogen activators in therapy will be discussed and further characterization of the new molecules will-be presented.
PROSTAGLANDIN E1 REDUCES ENDOCARDIAL PLATELET DEPOSITION AND INCREASES LEFT VENTRICULAR EJECTION FRACTION (LVEF) IN PATIENTS WITH CARDIOMYOPATHY (CMP)
Authors: Sinzinger H , Glogar H , Weidinger H , Sochor H , Auinger H , Weissel M , Fitscha P , Bergmann H , Granegger Susanne
Published: August 2018

It is well known, that patients suffering from CMP exhibit a significantly enhanced thromboembolic risk. Thus, we examined in these patients platelet function (f3TG, PF4, TXB2-serum and plasma) and kinetics after 111-In-oxine sulphate (100μCi) labelling. In a total of 67 patients (50o,17g; 29-66a with a LVEF of < 30%) the following findings have been obtained.

During γ-camera imaging and by engymetric monitoring using a portable detector system (Novo Int. Danmark) CMP-patients show an enhanced platelet deposition over the heart. Platelet half-life (1: 100-115h) is extremely shortened (< 50hrs). Prostaglandin (PG) E1 (prostavasin; α1-cyclodextrin PGE1; Sanol-Schwarz GmbH, Monheim, FRG) administered i.v. in a dose range of 5-50 ng/kg/min causes a significant (p< 0,01) increase in LVEF of 20-95% dose-dependently as measured by means of radioisotope technique.

Furthermore, a diminution in the local platelet uptake over the heart starting at the 2nd day of continuous PGE1-infusion can be monitored.

It is hypothetized, that PGE1 renders the endocardium less thrombogenic and increases LVEF by vasodilatation and decreasing the afterload. As PGE1 is metabo-lically degraded to a major extent during the first lung passage, the data after i.v. application are at least suggestive, that more stable PGE1-derivatives may contribute to this effect.

It is therefore concluded, that PGE1 in a safe dose of 25ng/kg/min may appear in the future as an optimal long-term therapeutic agent in CMP-patients offering the benefit of significantly enhanced LVEF and decreased platelet deposition, which should result in a decreased thromboembolic risk in these patients.
DYNAMICS AND LOCALIZATION OF PLATELET DEPOSITION ON A SYNTHETIC VASCULAR GRAFT: CONTINUOUS IMAGING
Authors: Badimon J J , Badimon L , Galvez A , Camunas J , Fuster V
Published: August 2018

The in vivo dynamics of thrombus formation have not been extensively studied, mainly due to technical limitations. We assessed the dynamics and localization of platelet deposition on a prosthetic vascular graft for the first 24 hours after implantation in swine, with continuous monitoring during the initial 6 hours, and the effect of heparin. Polytetrafluoro-ethylene (PTFE) grafts (5cm. L × 0.5 cm. ID) were inplanted in one of the common carotids of 13 normal pigs; 8 received iv heparin (150uAg) perioperatively. 111 In-labelled autologous platelets were injected 5 min before reperfusion of the graft. From 10 min to 24 hrs after unclamping the vessel sequential gamma camera images of the neck were taken and stored in an on-line computer. Pinpoint analysis of the platelet deposition was performed by creating seven regions of interest of 5 × 5 pixels over both graft and contralateral carotid territories. We obtained the ratio of the 111 In-activity in each region of the graft, including both anastomoses, with respect to its contralateral homologous region. The ratios differed along the graft in both groups of animals, with maximal values at the anastomosis. Peak ratios were reached within 1 to 3 hrs, and were significatively lower in heparinized pigs (anastomosis: 1.95±0.36; graft: 1.3±0.66) than in rion-heparinized-pigs (anastomosis: 3.23±0.66; graft: 2.16±0.41; p<0.05). Heparinized pigs showed a progressive decrease of the ratios up to 24 hrs. In contrast, platelet deposition in non-heparinized-pigs continued up to 6 hrs. Patency at 24 hrs was 88% in heparinized-pigs versus 20% in non-heparinized-pigs. We conclude that computer assisted pinpoint analysis of platelet deposition may help to a better understanding of the thrombotic process differentiating platelet-graft interaction from platelet anastomosis interaction. The deposition of platelets and graft patency is strongly influenced by the stabilizing effect of procoagulant moieties, and the presence of the anastomosis (release of vessel wall procoagulant and platelet activating products and induction of blood flow disturbances) induces localized activation and deposition of platelets.