bluuebunny/crossref_metadata_2025 huggingface dataset:
PLATELET ADHESION TO HUMAN AORTIC FRAGMENTS
Authors: Pintigny D , Wautier L J , Andreassian B , Caen P J
Published: August 2018

Due to the fact that the adhesion of platelet aggregates to microdamaged sites in the vascular endothelium holds a critical step in the pathogenesis of thrombotic and sclerotic plaques, we investigated the adhesion of platelets to arteritic vessel wall. Aortic samples were obtained from patients with peripheral vascular disease who required a prosthetic replacement in their aortic bifurcation. Aortic fragments were collected into buffer and tested within 24 hours after collection. The adhesion of normal platelets to the vessel fragment and the prostacyclin (PGI2) production were tested in 11 cases. Washed platelets from normal subjects were labeled with (51Cr)-sodium chromate, and incubated with aortic fragment or collagen. Non adherent platelets were separated by gel filtration through a Sepharose 2B column. Synthesis of PGI2 by the same aortic fragments was measured as 6-Keto-PGF1α a by radioimmunoassay after stimulation by thrombin (1 u/ml). In one case synthesis of PGI2 was high (80.9 ng/g dry-weight) and platelet adhesion was low (38.0%); in the other cases when PGI2 level was between 0.8 and 19.3 ng/g the adhesion was (a) low in 4 cases (0.7-40.3%) or (b) high in 6 (66.7-95.9%). In comparison adhesion to collagen performed in parallel was 78.5-95.7%. No correlation was found between platelet adhesion and the capacity to synthetize PGI2. These results suggest that in pathological conditions, when the endothelium is abnormal, platelet adhesion is not exclusively dependent on the PGI2 synthesis. In addition, this technique offers a quantitative test to measure the vessel wall thrombogenecity and its possible modulation by therapeutic agents.
THROMBOGENICITY OF THE VESSEL WALL AFTER SINGLE AND REPEATED INJURY
Authors: Pasche B , Swedenborg J , Ljungqvist A
Published: August 2018

Departments of Experimental Surgery and Pathology, Karolinska HospitalStockholm, Sweden Injury to the endothelial lining of the vessel wall gives rise to increased thrombogenicity but fibrin formation is only seen after repeated injury

The purpose of the present study was to simultaneously study the appearance of thrombin enzymatic activity and morphological changes on the injured vascular surface after primary and repeated injury. Endothelial injury was caused by balloon catheter in rabbits. The animals were sacrificed and the aorta was excised, inverted and mounted on a plastic rod. Thrombin enzymatic activity was measured on the surface by exposing it to either a chromo-genic substrate (thrombin$) or fibrinogen (thrombinp). In the latter case generation of fibrinopeptide (FPA) was measured. Standard light microscopic procedures were also performed After injury no endothelial cells were seen. Thickening of the internal elastic lamina and accumulation of granulocytes and platelets was also noted. Thrombin^ and thrombinp was detected on the surface. One week after injury the vessel wall had healed but the endothelial . cells were metachromatic and had numerable mitoses. No thrombin activity exceeding the control was seen. After repeated injury thrombins and thrombinp were again demonstrated but the fraction constituting thrombinp was larger than after primary injury. Microscopy at this time showed severe changes including vacuolisation

The capacity of the surface to inhibit thrombin in vitro was also studied and it was found that this capacity was lower after repeated injury particularly in the case of thrombinp

It is concluded that injury of the endothelium gives rise to appearance of thrombin enzymatic activity. After repeated injury proportionally more thrombinp is found, which may explain why fibrinogen is more easily demonstrated on the surface after repeated injury
PLASMA FIBRONECTIN IN ACUTE LEUKAEMIAS AND DURING STREPTOKINASE THERAPY
Authors: Bykowska K , Lapaciuk S , Janczarski M , Wegrzynowicz Z , Kopec M
Published: August 2018

We have previously shown that digestion of fibronectin (FN) by trypsin, kallikrein and plasmin influences strongly the FN quantitation by electroimmunoassay (EIA) and immunoturbidimetric assay (ITA). Proteolytic degradation led to an overestimation of FN by EIA but to a decline of results in ITA (Thromb.Haemostas., 53:377, 1985). In this study plasma FN was determined in parallel by EIA and ITA in adult patients with acute leukaemia prior to chemotherapy and in patients treated with SK for DVT. It has been assumed that in leukaemias leukocytic proteases can degrade FN. In 24 control subjects, mean values of plasma FN determined by EIA (285 ± 61 mg/1) and by ITA (268 ± 61 mg/1) did not differ. In contrast, significantly higher values were found by EIA (268 ± 100 mg/1) than by ITA ( 214 ± 65 mg/1, p < 0.01) in 38 patients with acute myeloid leukaemia (AML) and in 12 with blast crisis (BC) in chronic granulocytic leukaemia (245±73 mg/1 and 182 ± 46 mg/1 respectively, p < 0.05). Difference in results of two assays in 10 patients with acute lymphoblastic leukaemia (ALL) was not significant. A decrease in plasma FN when compared with controls was detected only by ITA but not by EIA in AML and BC (p < 0.001). The results of two immunoassays showed a high correlation in control group (r = 0.940) and in ALL (r = 0.838), lower in BC (r = 0.570) and poor in AML (r = 0.163). Discrepant were also results of parallel FN determination by EIA ai]dTJTA in 4 patients treated with SK. Higher values of EIA than of ITA could indicate plasmin mediated FN proteolysis. We have not confirmed recent reports on a frequent occurrence in leukaemias of slowly migrating peak of plasma FN complexes in two-dimensional crossed Immunoelectrophoresis. Such peaks were detected in only 3 of the 46 examined plasma samples. In conclusion the parallel FN determination by EIA and ITA can provide an ’ indirect evidence for the occurrence of proteolytic degradation of plasma FN in vivo.
FIBRONECTIN IS REQUIRED FOR PLATELET ADHESION AND FOR THROMBUS FORMATION ON SUBENDOTHELIUM AND COLLAGEN SURFACES
Authors: Bastida E , Escolar G , Castillo R , Ordinas A , Sixma J J
Published: August 2018

Fibronectin (FN) plays a role in several adhesion mediated functions including the interaction of platelets with subendothelium.We investigated the role of plasma FN in platelet adhesion and platelet thrombus formation under flow conditions.To do this we used two different perfusion models:1)the annular chamber with α -chymotrypsin-treated rabbit vessel segments and 2)the flat chamber with coverslips coated with fibrillar purified human collagen type III.Perfusates consisted of washed platelets, and washed red blood celIs,suspended in normal or FN-depleted plasma.Perfusions were carried out for 10 min at shear rates of 300 or 1300 sec™1 Platelet deposition and thrombus dimensions were morphometrically evaluated by a computerized system. We found that depletion of plasma FN significantly reduced the percentage of total coverage surface and percentage of platelet thrombus, at both shear rates studied, and in both perfusion systems (p < 0.01)(p < 0.01).The dimensions of the platelet thrombi formed in perfusions at high shear rate were also significantly reduced in perfusions carried out with FN-depleted plasma.(p < 0.01). Addition of purified FN to FN-depleted perfusates restored all the values to those measured in the control perfusions.These results indicate that, in addition to supporting platelet adhesion to the subendothelium and to fibrillar collagen, FN contributes to platelet thrombus formation under flow conditions.
PLATELET ADHESION AND THROMBIN DEPENDENT AGGREGATION OF PATIENTS WITH AN UREMIC BLEEDING TENDENCY
Authors: Zwaginga Jaap J , de Groot Philip G , Sixma Jan J
Published: August 2018

Five patients with chronic renal insufficiency (CRI) presented a Simplate bleeding time of > 30’, two patients had normal bleeding times (< 9’)- Blood was collected before standard hemodialysis into 19 mM citrate (plasma concentration). It was circulated fojr 5’ through an annular perfusion chamber at a shear of 1300 s™1 over inverted umbilical artery segments. CRI blood’s hematocrit was raised to .3 by adding their own RBC’s. Control whole blood perfusates with Ht .3 were made by addition of their own plasma. After perfusion platelet adhesion on the artery was evaluated by microscope, corrected for platelet count of the perfusate and given as percentage surface covered. Control donors showed a 37.4 ± 5.2% coverage not different from ‘bleeding’ patients 38.0 ± 4.5% ’non bleeding’ CRI patients: 32.3 ± 3.9. We also perfused blood of three ’bleeding’ CRI patients in a new thrombus forming system. In a rectangular perfusion chamber (J Lab Clin Med 1983, 522-532) blood anticoagulated with low molecular weight heparin (Fragmin1, Kabi Vitrum) was circulated over tissue factor containing matrix of 43-phorbol 12-myristate 13-acetate perturbed endothelial cells. Locally formed thrombin stimulated platelet aggregation on this matrix. Aggregation was expressed as percentage of spread platelets covered with aggregates. Perfusions with the following perfusates were performed: whole blood of controls (WBc) and patients (WBp), CRI platelets with normal plasma and RBC’s (A), CRI plasma with normal platelets and RBC’s (B) and normal platelets with normal plasma and RBC’s (C).

Platelet adhesion of CRI whole blood is not defective, aggregation, however, is. Uremic platelets in normal plasma may have an adhesion defect (A). The defective aggregation caused by uremic plasma (B) seems to be corrected for uremic platelets in normal plasma (A).
EFFECT OF DEXAMETHASONE ON ENDOTHELIAL EXTRACELLULAR MATRIX FORMATION AND ON ITS REACTIVITY TO PLATELETS IN A PERFUSION SYSTEM
Authors: Samaden A , Piovella F , Vigotti M , Bendotti C , Fregoni V , Ricetti M M , Montecchio G P
Published: August 2018

We previously demonstrated that dexamethasone increases fibronectin (FN) expression by human umbilical vein endothelial cell (HUVEC) as well as its incorporation in the extracellular matrix (EM). FN is primarily involved in platelet/vessel wall interactions as well as in cell attachment to substrates. The in vivo anti-haemorragic effect of glucocorticoids could therefore be related either to a better spreading and attachment of endothelial monolayer to the basement membrane or to an increase of vessel wall reactivity to platelets. To further investigate this, we studied the effect of dexamethasone treatment on EM reactivity to platelets in an in vitro perfusion system. Second passage HUVEC were seeded on glass coverslips at a density of 105/cm2 and treated with dexamethasone 1.0 μM for 48 hours. EM were prepared by exposure to 0.1 M NH4OH for 30’. Coverslips carrying the EM were perfused with citrated whole blood in a flat perfusion chamber(*) at wall shear rates of 300 and 900 sec™1 for 5’. Platelet adhesion to EM was evaluated by a morphometric method. The number of platelet adhering to EM at both shear rates was significantly increased by dexamethasone treatment, (+51.9% at 300 sec™1 (p<0.001) and +24.3% at 900 sec™1 (p<0.01)). Our results demonstrate that dexamethasone increases endothelial EM reactivity to platelets possibly through an increase of FN deposition. This, together with FN effect on cell spreading and anchorage to EM, might be responsible for the antl-haemorrhagic action of glucocorticoids.

(*) K. Sakanassen et al . , J . Lab. Cl in . Med. 102:522, 1983
IS THE ENDOTHELIAL EXTRACELLULAR MATRIX THROMBOGENIC OR THROMBORESISTANT? EFFECT OF PREPARATION AND 13-HODE LEVELS
Authors: Buchanan M R , Bastida E , Aznar-Salatti J , de Groot P
Published: August 2018

It is generally thought that the extracellular matrix (ECM) is thrombogenic.However,one of us (MRB) has reported that the ECM is thromboresistant,and postulated that this was due to the release of endothelial cell (EC) 13-hydroxyoctadecadienoic acid (13-HODE) into the ECM. To test this possibility, we measured platelet adhesion (PLT ADH) onto cultured ECs and their ECMs exposed by 3 methods. We also extracted the ECMs for HPLC analysis of 13-HODE.PLT ADH was expressed as i)adhesion of 3H-adenine labelled platelets/mm2 of ECs or ECMs under static conditions, and ii) % surface^ area coverage measured morphometrically following 5"perfusion with citrated whole blood at 1300 sec-1 in the flat chamber.ECMs were prepared by removing the EC monolayers by freeze thawing , cellulose acetate stripping or NH4OH treatment. PLT ADH to ECs under static and flow conditions were 4700±240/mm2 and 0.1%, respectively, and were associated with 12,6± 1 pg of 13-HODE/mm2 of EC surface (M+SEM). Removal of the ECs by freeze thawing or stripping, resulted in a 18% and 25% increase in PLT ADH to the ECM,under static and flow conditions respectively, and a 80% decrease in ECM associated 13-HODE level. Removal of the EC by NH4OH resulted in a 380% and 770% increase in PLT ADH to the ECM in static and flow conditions. 13-HODE was undetectable.

These data support the hypothesis that 13-HODE released from ECs influences the ECM thrombogenecity, and indicate that the residual amounts of components present in the ECMs following EC removal is influenced by the method of ECM preparation.
THE “MALMO“ EPITOPE OF FACTOR IX: PHENOTYPIC EXPRESSION OF THE “VIKING“ GENE
Authors: Graham J B , Lubahn D B , Kirshtein J D , Lord S T , Nilsson I M , Wallmark A , Ljung R , Bosco J , Frazier L D , Ware J L , Lin S-W , Stafford D W
Published: August 2018

The epitope of a mouse monoclonal AB (9.9) which detects a Factor IX (F.IX) polymorphism in the plasma of normal persons (PNAS 82:3839, 1985) has been related to not more than 6 AA residues of F.IX by recombinant DNA technology. The same 6 residues define Smith’s polymorphic epitope (Am. J. Human Genet. 37:688, 1985 and in press). This region of F.IX contains the alanine:threonine dimorphism at residue 148 first suggested by McGraw et al. (PNAS 82: 2847, 1985) and established by Winship and Brownlee with synthetic DNA oligomers (Lancet in press). Using synthetic DNA probes, we have found that the DNA difference between positive and negative reactors to 9.9 is whether base pair 20422, the first pair in the codon for residue 148, is A:T or G:C. We can conclude that 9.9 reacts with F.IX containing threonine but not alanine at position 148.

The F.IX immunologic polymorphism-whose epitope we are referring to as “Malmo”-is, not surprisingly, in strong linkage disequilibrium with two F.IX DNA polymorphisms, TaqI and Xmnl. The highest frequency of the rarer Malmo allele in 6 disparate ethnic groups was in Swedes (32%); a lower frequency (14%) was seen in White Americans whose ancestors came overwhelmingly from the Celtic regions of the British Isles; it was at very low frequency or absent in Black Americans, East Indians, Chinese and Malays. A maximum frequency in Swedes and absence in Africans and Orientals suggest that the transition from A:T to G:C occurred in Scandinavia and spread from there. The history of Europe and America plus the geographical distribution of the rare allele lead us to suggest that this locus might be designated: “the Viking gene”.
EXPRESSION OF HUMAN FACTOR IX IN MAMMALIAN CELLS
Authors: Wha Lin Shu , Ware J , Roberts H , McGraw N , McAllister W , Stafford D
Published: August 2018

Human factor IX has been expressed in mammalian cells. A cloned factor IX cDNA missing the first 15 nucleotides of the 5’ end was modified by in vitro mutagenesis to restore the missing codons and add the translation consensus sequence, CCACC, proposed by Kozak to be optimal for translational initiation. Additionally, Bgl II and BamHI sites were added immediately upstream of the CCACC sequence for ease of portability of the fragment. This modified cDNA was inserted into a bovine papillomavirus (BPV) vector under the control of a mouse met alio thionein promoter. The constructed plasmid pBPV-IX was used to transfect a mouse fibroblast cell line C127. After 3 weeks, the transformed foci were isolated and the established cell lines were grown in the presence or absence of vitamin K. Media was collected at 3 day intervals and assayed for factor IX activity in a one stage clotting assay. A standard curve was constructed using purified human factor IX. Cells grown in the presence of vitamin K (3 mg/L) exhibited an activity equivalent to 350 ng/ml of factor IX in the cell media; no (less than 3 ng/ml) activity was detectable in the absence of vitamin K. A monoclonal antibody column specific for the Ca++ dependent form of human factor IX allowed the isolation of approximately 7 ug of purified factor IX from approximately 100 ml of culture medium. Western blot analysis of the purified factor IX revealed 2 protein bands which reacted with a goat anti-human factor IX antibody as well as a human specific monoclonal antibody. One of the immunoreactive bands migrates with authentic human factor IX and the other migrates slower. This expression system provides a convenient way to produce suitable amounts of factor IX and mutated factor IX protein for functional analyses.
MYOCARDIAL SALVAGE BY COMBINED TREATMENT WITH RECOMBINANT SINGLE-CHAIN UROKINASE-TYPE PLASMINOGEN ACTIVATOR AND RECOMBINANT HUMAN SUPEROXIDE DISMUTASE IN A CANINE CORONARY THROMBOSIS MODEL*
Authors: Finke U , Schneider J , Friderichs E , Flohé L , Giertz H
Published: August 2018

Recanalization of thrombotic coronary occlusion with fibrinolytic treatment is a promising approach to salvage jeopardized ischemic myocardium. However, the success of thrombolytic treatment of myocardial infarction may be curtailed by the risk inherent to reperfusion. Cell damage upon reoxygenation after an ischemic period is tentatively attributed to the formation of oxygen-derived free radicals. Improved myocardial salvage is therefore expected from coadministration of a free radical scavenger and fibrinolytic treatment. We tested this hypothesis in a canine model of left anterior circumflex coronary artery (LCX) thrombosis. Thrombolysis was achieved with the fibrin- selective single-chain urokinase-type plasminogen activator of recombinant origin (r-scu-PA). As enzymatic scavenger of oxygen radicals recombinant human superoxide dismu-tase (r-HSOD) was used. The three experimental groups were: group I (n=4) did not receive any treatment after LCX thrombosis; in group II (n=9) at 100 min after LCX thrombosis r-scu-PA (20 μg/kg/min i.v. for 30 min) was infused; dogs in group III (n=8) received concomitant treatment with r-scu-PA and r-HSOD (10 mg/kg i.v. for 60 min). Infarct size as percent of the risk zone was 38.2 ± 4.1 in group I, 25.3 ± 3.7 in group II (p ≤ 0.05 vs group I) and 14.9 ± 3.2 in group III (p ≤ 0.05 vs group II). Incidence of reperfusion arrhythmias and increase in plasma CK were significantly diminished by r-HSOD when compared to dogs receiving r-scu-PA only. There were no significant differences in hemodynamic parameters between the groups. In conclusion, in terms of infarct size reduction, suppression of reperfusion arrhythmias and attenuation of intracellular enzyme release the combined treatment with r-scu-PA and r-HSOD yielded a significant higher myocardial salvage versus thrombolytic treatment alone in a canine LCX thrombosis model.

* This work is part of the doctoral dissertation of Miss U. Fincke
INFLUENCE OF RECOMBINANT PRO-UROKINASE ON THE HEMOSTATIC SYSTEM IN PATIENTS WITH ACUTE MYOCARDIAL INFARCTION
Authors: Schmitz-Huebner U , Ostermann H , Mathey D G , Schofer J , Diefenbach Ch , Erbel R
Published: August 2018

Recombinant unglycosylated pro-urokinase (recombinant single chain urokinase-type plasminogen activator, CG4509) was given to twelve patients (pts.) with acute myocardial infarction as a 20 mg bolus followed by a 60 mg intravenous infusion (iv.inf.) over 1 hour and to twelve pts. as a 10 mg bolus followed by a 30 mg iv.inf. over 1 hour. Reperfusion was angiographically confirmed in 9/12 pts. with the higher dose and in 6/12 pts. who obtained the lower dose. Different parameters of hemostasis were determined before administration, 30 min after the beginning of inf.,at the end of inf., 60 min thereafter and 6-12 hours afterwards .

The most significant systemic changes were observed 60 min after the end of inf. when the following mean values ± SEM were determined (pre-inf. values in brackets):

Fibrinolytic activity in plasma determined on fibrin plates was highest in both groups 30 min after the beginning of inf. and hardly measurable 60 min after the end of inf. No bleeding complications were observed. Based on these results,a randomized double-blind multicenter trial was started to study the effects of 80 mg CG 4509 versus 1.5 million U streptokinase iv.
THE EFFICACY AND RELATIVE FIBRIN SELECTIVITY OF PROUROKINASE IN PATIENTS WITH ACUTE MYOCARDIAL INFARCTION
Authors: Loscalzo J
Published: August 2018

The use of thrombolytic agents in acute myocardial infarction has gained widespread acceptance as an important early therapeutic option. Acute coronary thrombosis has been found in approximately 80% of patients with acute infarction and the use of standard thrombolytic agents opens these occluded vessels in most cases. Unfortunately, in many individuals standard agents also produce a systemic lytic state with its attendant hemorrhagic complications. Prourokinase (PUK) has been shown to be a relatively fibrin selective thrombolytic agent in vitro owing to its localized conversion to urokinase at the clot surface. Because of this desirable property, we studied the efficacy and selectivity of PUK in vivo in 19 patients with acute myocardial infarction. Each of these patients was documented by angiography to have a totally occluded infarct-related artery. Each patient was treated within six hours (range: 2 to 5.8 hours) of the onset of symptoms with 62.5 mg of PUK derived from the human kidney cell line, TCL598, infused intravenously over 90 minutes. Complete vessel patency was achieved in nine patients within 61 ± 19 minutes of the start of the infusion without apparent hemorrhagic complications. We evaluated the effect of PUK on fibrinogen, on nonspecific fibrinogen degradation products (FDP) the the fibrinogen/fibrin I peptide, Bβ 1-42, as well as on the specific fibrin degradation products, D-dimer (XDP) and the fibrin II peptide, Bβ 15-42. Values for these parameters measured before and at the end of the 90-minute infusion of PUK are given as the mean ± S.E.M.

We conclude that PUK is an effective agent with which to achieve coronary thrombolysis and that at the doses used in this study, it aDDears to be relatively fibrin selective.
ANTITHROMBOTIC EFFECTS OF TISSUE-TYPE PLASMINOGEN ACTIVATOR AT PHYSIOLOGICAL BLOOD LEVELS
Authors: Witt W , Baldus B , Donner P
Published: August 2018

Effective thrombolysis in human patients and experimental animals by tissue-type plasminogen activator (t-PA) usually requires t-PA plasma levels in the microgram range. Compared to that physiological plasma levels of t-PA are about 100 - 1000 times lower. To investigate the effects of t-PA at physiological blood levels rat studies were performed in vitro and in vivo employing highly purified recombinant single-chain t-PA (sct-PA: 500,000 IU/mg).

t-PA activity in rat whole blood as assessed by dilute blood clot-lysis time (DBC-LT) was increased by addition of sct-PA as low as 3 ng/ml (20 % decrease in DBC-LT). Injection of brady-kinin 10, 100 and 1000 μg/kg i.v. shortened DBC-LT to 54, 23, and 10 % of controls corresponding to the effect of about 10, 30, and 100 ng/ml sct-PA added in vitro. Infusion of sct-PA 15 - 450 μg/kg/h i.v. shortened DBC-LT ex vivo dose-dependently by 20 - 90 % at steady state levels (n = 5). In the same dose range sct-PA inhibited thrombus formation along a silk thread introduced into an arteriovenous shunt in anaesthetized rats. The reduction in thrombus dry weight was dose-dependent amounting to 33 - 67 % of preapplication values (n = 5 - 8) at 15 - 450 μg/kg/h i.v. sct-PA. Already 50 μg/kg/h sct-PA corresponding to a sct-PA activity of about 15 ng/ml displayed a significant (a = 0.05) effect in this model.

The results of this study suggest that t-PA present at physiological resting or activation (bradykinin) levels during acute clot formation may have potent antithrombotic efficacy. This study provides further evidence for the importance of a balance coagulation-fibrinolysis which can be influenced on both sides towards thrombophilia as well as to achieve antithrombotic therapy, e.g. by elevating plasma fibrinolytic activity with low-dose t-PA treatment or with drugs which stimulate the endogenous fibrinolytic potential.
A COMPARISON BETWEEN FIBRINOLYSIS OF FRESH AND AGED CLOTS OR THROMBI BY rt-PA IN VIVO, EX VIVO AND IN VITRO
Authors: Nauland U , Haarmann W , Müller T H , Eisert W G
Published: August 2018

In view of the therapeutic applications of rt-PA it is of interest to investigate whether there is any difference in the lysability between fresh and aged thrombi. The efficiency of fibrinolysis by rt-PA was studied in 3 different ways: In vivo, by measuring the thrombus weight of fresh (1 h) or aged (24 h) thrombi in the carotid artery of rabbits which had been treated with rt-PA (0.4 mg/kg) or saline for 1 h. Ex vivo, by measuring I125-release of in vivo fresh (1 h) and aged (24 h) thrombi (labelled with I125-fibrinogen) suspended in vitro in plasma containing rt-PA (1 μg/ml) ; the thrombi were formed in the jugular vein and the carotid artery of each rabbit. In vitro, by measuring I125-release of fresh (1 h) and aged (6 or 24 h) human native whole blood clots, PPP-clots, PRP-clots and squeezed PPP-clots which were formed and lysed in vitro with rt-PA (1 μg/ml) . In vivo as well as ex vivo rt-PA lysed fresh (1 h) thrombi much better than aged (24 h) thrombi. This difference was more pronounced immediately after the onset of fibrinolysis, but decreased with time. However, in vitro relatively little difference was observed in fibrinolysis efficiency between fresh (1 h) and aged (6 or 24 h) clots; fibrinolysis of these clots was decreased (PPP > whole blood > PRP) with increasing clot retraction, which was almost complete within 1 h. This result was also confirmed when PPP-clots were “retracted” by simply squeezing them just before lysis. Therefore we conclude that a considerable difference in lysis efficiency between fresh and aged thrombi was only observed when thrombi were formed and aged in vivo. This difference was less pronounced with increasing fibrinolysis time.
MYELOPROLIFERATIVE DISEASE CHARACTERIZED BY THROMBOSIS, BLEEDING AND PLATELET DYSFUNCTION IN MICE INJECTED WITH THE POLYOMA MURINE LEUKEMIA VIRUS (PyMLV)
Authors: Di Minno G , Fusco A , Portella G , Cerbone A H , Iride C , Tajana G , Russo O , Mattioli P L
Published: August 2018

After i.p. injection of PyMLV, NIH/OLAC mice showed thrombi in tail veins, ears, muscles and nesenterium together with thrombi and hematomas of subcutaneous tissues. This was followed by infarctions of lungs, brain and heart, that caused death of the animals. Laboratory evaluations of the infected mice showed normochromic anemia, mild thrombocytosis and marked defects in the aggregation and in the secretion of ATP from platelets exposed to AJP, collagen, thrombin or A23137. About 10% of cells present in the bone marrow was formed by blasts; 20% by multinucleated cells identified as megakariocytes (M) by peroxidase and acethyl-cholinesterase staining, and the vast majority of the other cells by entities belonging to all stages of maturation of the myeloid lineage. Hybridization experiments showed that the blasts present in the bone marrow were the only cells in which viral replication takes place. Maturation of M in the bone marrow was completely normal, and as for M from non-infected mice, proliferation and maturation in vitro was dependent on the presence of interleukin 3. Finally, studies in other strains of mice showed that the Fv-2 locus is involved in the pathogenicity of PyMLV in NIH/OLAC mice. Ne conclude that, in addition to its obvious pathophysiological significance, the myeloproliferative disease that occurs in mice after i.p. injection of PyMLV, can serve as an important probe for understanding basic events leading to bleeding and thrombosis.
THE THIENOPYRIDINE PCR 4099 INHIBITS THE ADP AGGREGATION PATHWAY OF HUMAN PLATELETS BY INTERFERING WITH THE BINDING OF FIBRINOGEN TO THE GLYCOPROTEIN IIb-IIIa COMPLEX
Authors: Gachet C , Stierlé A , Bouloux C , Maffrand J-P , Cazenave J-P
Published: August 2018

The thienopyridine, PCR 4099, is a synthetic structural analog of ticlopidine. After oral administration in man, it prolongs the bleeding time (BT) and inhibits ADP-induced aggregation. The aim of the study was to evaluate the effects of oral administration of 200 mg per day PCR 4099 to 10 human volunteers for 7 days on primary hemostasis and to study the mechanism of inhibition of the drug on the ADP-fibrinogen-GPIIb-IIIa pathway of aggregation. BT (measured by a Simplate device) was 4-8 min before treatment and 30 min after 7 days of treatment. Platelets were washed and resuspended in Tyrode’s buffer containing apyrase and 0.35 % human albumin. Washed platelet suspensions were used at 37°C for aggregation and fibrinogen binding studies. Human fibrinogen was purified by successive ether precipitation and gelatin affinity chromatography to remove fibronectin. Fibrinogen was pure bv SDS-PAGE and > 95 % clottable by thrombin. It was labeled with 125I by the Iodogen method. The binding of 125I-fibrinogen to intact washed platelets exposed to ADP or thrombin was measured after centrifugation at 11 ,000 g for 1 min in the presence of 131I-human albumin as a space marker. The membrane GPIIb-IIIa complex was examined by crossed immunoelectrophoresis (CIE) in the presence of rabbit anti-human platelet antiserum. The prolonged administration of PCR 4099 inhibited almost completely platelet aggregation induced by 0.5 to 10 μM ADP. Although the effect of ADP on aggregation was blocked at high concentrations, PCR 4099 did not modify ADP-induced shape change. Only the effects of low concentrations of thrombin (< 0.05 U/ml) were inhibited by PCR 4099 administration. The binding of 125I-fibrin0gen was reduced by 50 to 90 % when platelets were stimulated by 5 μM ADP or by 0.05 U/ml thrombin. PCR 4099 did not modify the pattern of immunoprecipitates as revealed by CIE. In particular the GPIIb-IIIa complex was not dissociated and its electrophoretic mobility was not changed. In conclusion, PCR 4099, which is more potent than ticlopidine in man, inhibits specifically the ADP aggregation pathway by interfering with the binding of fibrinogen to the GPIIb-IIIa complex in platelets having undergone shape change.
INTERACTIONS BETWEEn PGE2 AND INHIBITORS OF PLATELET AGGREGATION THAT ACT THROUGH cAMP
Authors: Gray S J , Heptinstall S
Published: August 2018

PGE2 has a biphasic effect on platelet aggregation with low concentrations of the prostaglandin potentiating aggregation and high concentrations inhibiting it. In this investigation we have studied the interaction of PGE2 with agents that inhibit platelet aggregation through an effect on cAMP. The agents chosen raise the level of cAMP in platelets by different mechanisms: PGI2, PGD9 and adenosine combine with specific surface-located receptors and stimulate adenylate cyclase (AC) via a guanine nucleotide-binding protein (GNBP), forskolin stimulates AC directly, and AH-P 719 and DN 9693 inhibit cAMP phosphodiesterase (PDE). ADP-induced platelet aggregation was measured in platelet-rich plasma and cAMP was measured in platelets labelled with 3H-adenine.

PGE2 alone potentiated platelet aggregation at concentrations from 10™8 -10™6 M and inhibited aggregation at 10™5M. PGE2 did not reduce cAMP levels at any concentration and increased cAMP levels at concentrations > 10™6 M, profcably by stimulating AC.

PGI2 (10™9 -10™8 M), PGD2 (10™7 -5×10™6 M) and adenosine (8×l0™5-2×10™4 M) increased the level of cAMP in platelets and inhibited aggregation these changes were reversed by low concentrations of PGE2 (10™8-10™6M).

Forskolin (5×10™6-2.5×10™5M), AH-P 719 (10™7-10™5M) and DN 9693 (5×10™6 -10™5M) increased the level of cAMP in platelets and inhibited aggregation. However, PGE2 did not reverse the inhibitory effects of these particular agents. In contrast, PGE2 potentiated the effects of the agents at all the concentrations of PGE2 that were tested (10™8-10™5M).

The different results obtained with PGE2 in combination with agents that act via surface-located receptors compared with agents that stimulate AC directly or act through PDE, suggest that PGE2 may potentiate platelet aggregation by acting at a point between the platelet receptor and AC i.e. GNBP.

PGE2 is one of the major prostaglandins synthesised by human microvascular endothelial cells and interstitial cells of the renal medulla. Since it reverses the inhibitory effects of some AC stimulators but adds to those of PDE inhibitors, the latter may have greater potential as anti-thrombotic agents in the micro-circulation and intra-renal circulation.
DN-9693: A PHOSPHODIESTERASE INHIBITOR WITH A PLATELET MEMBRANE EFFECT
Authors: Ball J , Greaves M , Jackson C , Peel J , Preston F E
Published: August 2018

We have examined the effect of DN-9693 (piperidinyl - imidazo - quinazoline: Daiichi Seiyaku, Japan) a water soluble phosphodiesterase inhibitor on platelet aggregation, secretion and thromboxane B2 (TXB2) production. In platelet rich plasma and at concentrations of 2uM and 5uM the drug significantly inhibited aggregation induced by adenosine diphosphate, collagen and sodium arachidonate. TXB2 production and release of adenosine tri-phosphate and 14C 5-hydroxytryptamine were also significantly inhibited by the drug. Cyclic adenosine mono-phosphate accumulation was enhanced. Inhibition of ristocetin induced platelet agglutination was an unexpected finding and further experiments were undertaken to explore this. These suggested no specific effect against a plasma factor (von Willebrand factor) and reduced expression of the platelet membrane glycoprotein Ib was implicated. To investigate this further we examined the effect of DN-9693 on the binding of a monoclonal antibody (McAb) to platelet membrane glycoprotein lb (AN51). This was assessed by a FACS IV flow cytofluorimeter utilising a goat anti-mouse fluorescein isothiocyanate (FITC) labelled secondary antibody. Similar experiments were also performed with McAbs to the membrane glycoprotein complex IIb/IIIa (M148) and also to glycoprotein IIIa (C17). In platelet rich plasma, at concentrations which have been shown to inhibit aggregation, DN-9693 significantly reduced the mean fluorescence intensity of the cells coated with McAb AN51 in a dose related manner. This strongly suggested a drug effect against the glycoprotein Ib receptor site. Also, the drug appeared to enhance the binding of McAb C17 to glycoprotein IIIa. This study indicates that in addition to potent phosphodiesterase inhibitor activity, DN-9693 causes a platelet surface membrane change which is associated with reduced expression of membrane glycoprotein Ib.
NEW INSIGHTS INTO THE ANTIPLATELET ACTIVITY OF FORSKOLIN: ROLE OF PLASMA ADENOSINE AND SPECIES DIFFERENCES
Authors: Agarwal Kailash C
Published: August 2018

Forskolin stimulates adenylate cyclase by interacting with the catalytic subunit and inhibits platelet aggregation. This inhibition is greatly potentiated by adenosine (Ado) which stimulates adenylate cyclase through membrane-bound Ado receptors. Forskolin is 2-4 fold more potent as an inhibitor of collagen-induced rat platelet aggregation as compared to human platelets (IC50 values, in rat PRP, 0.5-0.8 μM; in human PRP, 1.5-2 μM). However, if the blood is pretreated with adenosine deaminase (ADA), an enzyme that degrades Ado to inosine, the inhibitory action of forskolin is greatly reduced producing similar effects both in human and rat PRPs (IC50, 2-3 μM) and whole blood (IC50, 4.6 μM). Both 5’-methylthioadenosine (MTA, 50-100 μM), an antagonist of Ado receptors, and 2’,5’-dideoxyadenosine (DDA, 100 μM), an inhibitor of adenylate cyclase, reverse the inhibition of platelet aggregation in rat PRP, whereas, no reversal is seen in human PRP. When Ado in the rat plasma is degraded by ADA pretreatment, DDA or MTA shows no reversal as seen in human PRP. The inhibitory action of forskolin (1-2 μM), which is only weakly inhibitory alone (<20%) in human whole blood, can be greatly potentiated (100% inhibition) by the inhibitors of nucleoside transport, dipyridamole (10 μM) or dilazep (2 μM). Only slight potentiation is seen in rat whole blood suggesting that rat plasma Ado levels are not affected significantly perhaps due to weakly active erythrocytic nucleoside transport system. Sato and Ui (In: Physiology and Pharmacology of Adenosine, Daly et al, Eds. Raven Press, 1983, 1-11), have shown that rat plasma contains much higher Ado levels (7.55 ± 0.51 pM) than human plasma (0.29 ± 0.08 μM). These studies demonstrate that plasma adenosine plays an important role in the forskolin antiplatelet activity which can be greatly potentiated in human whole blood by the clinically used drugs, dipyridamole and dilazep. (Supported by US PHS Grant CA 07340).
THE STRUCTURE OF PLATELET-REACTIVE SITES IN COLLAGENS
Authors: Barnes M J , Fitzsimmons C M , Morton L F
Published: August 2018

Collagen-induced platelet aggregation is an essential step in haemostasis and may be important in thrombosis, particularly that associated with the atherosclerotic plaque. We have located platelet-binding sites in collagen by fragmentation of the molecule with cyanogen bromide (CB) and measurement of the platelet aggregatory activity of the fragments following their renaturation to restore triple-helical configuration and polymerisation to introduce quaternary structure. We have found a high1y-reactive site in collagen type III located in the peptide α1(III)CB4 which was active at a concentration of less than 0.5μg/ml. The equivalent peptide from type I collagen α1(I)CB3 occurring in precisely the same location in the respective parent molecule (residues 403-551) and exhibiting a structure highly homologous to that of α1(III)CB4 was active only at concentrations higher than 200μg/ml. α1(I)CB7, the most active of the type I peptides, was able to cause platelet aggregation at around 5μg/ml whilst the equivalent type III peptide μ1(III)CB5 was inactive. Modification of specific amino acid residues indicated the importance of lysine in the activity of μ1(III)CB4 and of arginine in that of α1(I)CB7. Comparison of the structure of these peptides leads us to conclude that a reactive site comprises two basic residues, a specific distance apart, the conformation of one to the other dictated by collagen triple-helical configuration. One residue occurs in the sequence GlyPro(orHyp)LYS(orARG)GlyGlu, the other in GlyLYS(orARG)Pro(orHyp)GlyGlu. The lower reactivity of α1(I)CB7 relative to α1(III)CB4 can be attributed to the presence of two arginyl rather than lysyl residues and because the spacing of the two in CB4(G1y-LYS-Y-G1y-X-Y-G1y-X-LYS) represents a more favourable one than in CB7(Gly-X-ARG-Gly-X-Y-Gly-X-Y-Gly-ARG-Y).
THROMBOEMBOLISM PROPHYLAXIS IN SPINAL CORD INJURY: FIXED VS ADJUSTED DOSE HEPARIN
Authors: Green D , Cohn T , Filbrandt P , Ito V , Lee M Y , Press J , Vandenberg W C
Published: August 2018

We had previously estimated the incidence of deep vein thrombosis (DVT) in untreated spinal cord injury patients with complete motor paralysis to be 78% (Paraplegia 20:227, 1982). Therefore, we have begun to randomize patients to receive prophylaxis with either fixed dose heparin (5,000 u every 12 h subcutaneously) or adjusted dose heparin (mean dose, 13,890 u every 12 h). Treatment is started within 72 hrs of injury, and continued for 12 weeks. Nineteen subjects have received the fixed dose for 155 weeks, and 21 the adjusted dose for 153 weeks (p = N.S.). Patients have had daily clinical examinations for thromboembolism/bleeding, and weekly doppler flow and impedance plethysmography studies. All suspected DVTs have been confirmed by venography. The activated partial thromboplastin time (aPTT) with the fixed dose has average 26 ± 1.5s (S.D.) and with the adjusted dose, 39.6 ± 7.6s (p < 0.001). There have been 2 episodes of pulmonary embolism and 2 DVTs in the fixed dose group and 1 DVT in an adjusted dose patient whose aPTT never rose above 26s. The thromboses were noted after 2, 3, 3, 4, and 6 weeks of prophylaxis. Bleeding occurred in 2 patients on the adjusted dose regimen and required withdrawal of the heparin; no subject on the fixed dose bled. Our preliminary conclusion is that heparin prophylaxis significantly reduced the frequency of thromboembolism in spinal cord injury patients. While fewer and less severe thrombotic events have occurred in those receiving adjusted doses, the decrease has not yet reached statistical significance (x2 = 1.16). Furthermore, there appears to be a small, but definite risk of bleeding on this regimen. Thus, additional patients are being studied to determine the optimum use of heparin in this population.
THE CONTRIBUTION OF ENDOGENOUS UROKINASE (UK) AND TISSUE PLASMINOGEN ACTIVATOR (t-PA) TO SPONTANEOUS CLOT LYSIS IN PLASMA
Authors: Gurewich V , Emmons F , Pannell R
Published: August 2018

Spontaneous lysis of 125I-labeled clots was measured in order to study the relative contributions of pro-UK, t-PA and contact activation. Clots were made up from 0.3 ml plasma, platelet rich plasma (PRP) or 2% human fibrinogen (Kabi) and suspended in plasma (3 ml) prepared from blood freshly collected onto citrate. The clots were preincubated to inactivate any residual thrombin which could inactivate pro-UK. Antibiotics were added to suppress bacterial growth. Lysis was approximately linear and went to completion with plasma clots in 12-16 d. and with fibrinogen clots in 6-10 d. Spontaneous lysis was inhibited by aprotinin at >200 KlU/ml and by antibody to t-PA. When the latter was added at t 2-5 d., clot lysis was arrested at each time point suggesting that t-PA was predominantly responsible for the lysis and not just for its initiation. Addition of antibody to UK or immunodepletion of plasma had little effect on spontaneous lysis of plasma or of fibrinogen clots, but retarded lysis of PRP clots. Only after ≥20 ng/ml of pro-UK were added to plasma, was a dose-responsive acceleration of clot lysis observed. The addition of antibodies to t-PA inhibited clot lysis by the added pro-UK (20-80 ng/ml) and greatly prolonged the lag phase of clot lysis by higher concentrations of pro-UK (100-200 ng/ml). Contact activation by dextran sulfate (1-5 μM) had no effect on spontaneous clot lysis but potentiated the effect of > 20 ng/ml added pro-UK. It was concluded that 1) The UK content of resting plasma (3-5 ng/ml) contributes little to lysis of platelet-free clots. 2) The presence of physiological concentrations of t-PA is essential for lysis by endogenous UK and markedly potentiates fibrinolysis by added pro-UK (20-200 ng/ml). 3) For endogenous UK to contribute to fibrinolysis, some amplification of its effect by a cofactor other than t-PA seems to be required. The results indicate augmentation of endogenous pro-UK induced fibrinolysis by platelets and that contact activation stimulates clot lysis by added pro-UK.
CONTENTS OF PHYLLOQUINONE AND MENAQUINONE FAMILY IN SERUM AND FECES FROM HUMAN NEWBORN INFANTS
Authors: SHIRAHATA A , ASAKURA A , NAKAMURA T , YAMADA K
Published: August 2018

A sensitive and specific method for the determination of phylloquinone (PK) and menaquinone families (MK-n), between MK-4 and MK-10, was developed. Vitamin K (VK) was extracted from serum and feces with n-hexane-ether, and purified with silica gel column and short alumina column. Eluted VK was separated by high performance liquid chromatography using Cosmosil 5 Cie column with ethanol-water as a mobile phase. The separated VK was detected by a fluorometry after its reaction with ethanolic sodium boro-hydride in a reaction coil connected by one-line to a chromatographic column. Minimum detectable quantities of PK, MK-4 and MK-7 were 0.1, 0.1 and 0.15 ng/ml, respectively.

In normal adult serum, PK, MK-4, MK-5, MK-6, MK-7 and MK-8 were detected. The meaniSD values of PK and MK-n from normal adult serum were 3.0±1.4 (PK), 0.3±0.2 (MK-4), 0.9±0.4 (MK-5), 0.3 ±0.3 (MK-6), 3.8±1.4 (MK-7) and 0.3±0.2 (MK-8)ng/ml, respectively. On the other hand, VK was not detected in 19 umbilical blood samples, excluding 3 samples in which 0.7, 1.0 and 2.5 ng/ml of PK were detected. In 7 healthy newborn infants under 8 days of age, VK was not detected excluding 3 cases. Of these 3 cases, in one case, 3.16 ng/ml of PK was detected, and in the other two cases, 1.1 and 1.5 ng/ml of MK-7 were detected. In 11 infants at one month of age, PK ( 10 cases ) and MK-7 ( 5 cases ) were detected, and mean+SD values of PK and MK-7 in the detected group were 0.55±0.44 and 0.62±0.18 ng/ml, respectively. VK was not detected in plasma of infants with primary hemorrhagic disease of the newborn ( 4 cases ) and infantile vitamin K deficiency ( 2 cases ). With a wide variation of VK content in each samlpe, PK and all MK families were detected in feces from normal adults. On the other hand, only PK and MK-7 were detected in meconium collected from 6 normal newborn infants before initial feeding. The contents of VK in meconium were less than one percent of those in adult feces. These results indicate that VK supply from maternal side in utero and from intestinal flora after birth is very poor in newborn infants.
THE ACTION OF HEPARIN IN PLASMA
Authors: Coenraad Hemker H
Published: August 2018

Much is known about the influence of heparin on the interaction between coagulation proteases and antithrombin III. In the complex milieu of clotting plasma and a fortiori in platelet rich plasma many proteins compete for the heparin, the proteases are partly protected from AT III (-heparin) action and several antiproteases compete for one protease. Also the concentrations of the reactant vary in the course of the clotting process. This makes it difficult to obtain a clear insight in the action of heparin in plasma from studies on purified system only. We therefore developed a method that allows us a) to measure the breakdown constant of endogenously generated thrombin in plasma and to determine what part of it is due to bindingα2 macroglobulin, b) derive the time course of prothrombinase activity from the thrombin generation curve. This method allows us to study separately the action of heparin on prothrombinase and on thrombin.

Using this method we could show that: a) Classical heparin acts primarily on thrombin. Its action on other activated clotting factors is of secondary importance,b) The product activation of thrombin isespecially important in the intrinsic pathway, the thrombin dependent activation of factor VIII is the process that determines the onset of explosive thrombin formation. This explains why the APTT is much more sensitive to heparin than the PT is. c) The low molecular weight heparins that we. studied can be divided into two classes: the S type heparins, that, like unfractionated heparin, act practically only on thrombin and the P type heparins that do not act on thrombin but do inhibit prothrombinase. The synthetic pentasaccharide of Choay et al. is a typical example ofthis last group, d) We demonstrated a cooperative effect between thromboplastin and platelets that is mediated via thrombin.It is expressed as a shortening of the agphase of thrombin formation in plateletrich plasma, e) Heparin, but also other antithrombotic drugs inhibit the cooperative effect mentioned above. This suggests that this effect is a good candidate for the screening of antithrombotic drugs, f) The amount of thrombin formed in platelet rich plasma is hardly influenced by concentration of heparin that completely inhibits the formation of thrombin in platelet poor plasma. This is caused by the release of platelet factor 4. g) S type low molecular weight heparins can be conveniently standardized with a standard of classical heparin. P type low molecular weight heparins require a P type standard preparation.