bluuebunny/crossref_metadata_2025 huggingface dataset:
PROSTACYCLIN (PGI2) FORMATION IN RELATION TO ANAPHYLATOXIN C5a GENERATION DURING RABBIT ENDOTOXIN SHOCK
Authors: Rampart M , Bult H , Herman A G , Jose P J , Williams T J
Published: August 2018

Injection of endotoxin (LPS) in animals, a model for gram-negative septic shock, leads to intravascular activation of the complement system, and is one of the few conditions in which 6-oxo-PGF]CX and thromboxane (TX) B2 (non-enzymic metabolites of PGI2 and TXA2) can be detected in arterial blood. Previously we reported associations between complement activation, PGI2 biosynthesis and LPS-induced hypotension in rabbits. As C5a and C5adesArg trigger endothelial PGI2 formation in vitro, we have now measured the plasma levels of immunoreactive (ir) C5a in relation to generation of PGI2 and changes in arterial blood pressure in LPS shock. Pentobarbitone anaesthethized rabbits received LPS (E. coli 0111:B4, 0.5 mg/kg) or
saline via the marginal ear vein. A catheter in the left carotid artery was used to collect blood and to monitor mean arterial blood pressure (MABP). Platelet and leukocyte numbers, haemolytic complement titre (CH50), and plasma ir6-oxo-PGFioc , irTXB2 and irC5a were measured 15 min before and at different times after saline or LPS injection. LPS caused a dose- and time-dependent formation of irC5a in rabbit serum in vitro, predominantly via the classical pathway. LPS also activated complement in vivo, as indicated by about 20 % reduction of CH50 titre (measured after 3h) and a marked increase of arterial irC5a (20-120 ng/ml) in the first 2 to 5 min. After 30 min, irC5a had returned to baseline levels (< 2-5 ng/ml) and remained so up to 3h after injection of LPS. This irC5a peak correlated with a shortlasting initiation of PGI2 release (from < 20 pg/ml up to 550 pg/ml) and a drop in MABP (from about 95 mmHg to 50 mmHg) 2-5 min after LPS. None of these changes occurred after saline injection.

In conclusion, LPS activates complement in vivo with concomitant formation of C5a. This peptide may trigger -either directly or after phagocyte activation - endothelial PGI2 biosynthesis, leading to arterial hypotension. This is supported by the suppression of the initial rise of arterial ir6-oxo-PGF1α and hypotension in complement-depleted rabbits. Inhibition of C5a formation or activity may prove to be a meaningful approach to the treatment of septic circulatory shock.
HEPARIN BINDING TO ENDOTHELIAL CELLS POTENTIATES THROMBINSTIMULATED PGI2 PRoDUCTION
Authors: Bårzu T , Molho P , Cariou R , Tobelem G , Caen J
Published: August 2018

We studied the consequences of hepari n (H) binding to endothel iaI cells (EC) on their basal and thrombin-stimulated PGI2 production. Primary cultures of human umbilical vein EC were incubated with different H concentrations, in serum free medium for 5 hrs. The amount of 6-keto-pGF1α was measured in the medium after 5 hrs with an enzyme-linked inmunoassay. At all concentrations used (0.75 to 75 μg/ml) H did not alter the 5-hour basal production of PGI2 (control, 10.1 α 1.4 ng/106 cells; H 15 μg/m1 ; 10.8 ± 2.7 ng/106 cells). Basal or thrombin (0.1 U/m])-rtimulated (10 min) pGI2 production was then determined using EC bearing only bound heparin. The low, unstimulated PGI2 release (0.412 ± 0.04 ng/106 cells) was not significantly changed in the presence of bound H, but the thrombin-stimulated release was potentiated.

The KD for H binding to EC is 2.5 μg/ml. Thus at 3 μg/ml, half maximal saturation of binding sites and maximal potentiation of thrombin action were achieved. This concentration of bound H shifted the dose-response curve of thrombin induced PGI2 production to the left. Similar effect was obtained with half maximal saturating concentration of low molecular weight H (CY 222). Neither arachinodate nor LC4-induced PGI2 release were modified by H binding to EC, suggesting that potentiation is specific to thrombin. Since bound H was shown to not modify the high affinity thrombin binding to EC, the potentiating effect of bound H could related rather to interference with the specific mechanism of thrombin-stimulation of PGI2 production.
HIGH SUSCEPTIBILITY OF RABBIT, DOG, AND PIG PLATELETS TO SHEAR-INDUCED INJURY
Authors: Bauman J E , Joist J H , Vogler G , Sutera S P
Published: August 2018

In an attempt to develop an animal model for the study of the effects of fluid shear stress on platelet in vivo survival we examined the effects of repetitive short-duration (5 sec) and continuous prolonged (5 min) shear exposure in a cone-plate viscometer and Couette rotational viscometer on platelets (in citrated platelet-rich plasma) from humans, rabbits, dogs, and pigs. Comparable platelet aggregation (PAG = loss of single platelets) (18-64%) was observed with platelets from all species, associated with dense granule release, as a function of shear stress amplitude (25-50 dyn/cm2) under the conditions used. However, whereas with human platelets, little or no platelet injury (loss of LDH) was observed, appreciable platelet LDH loss was found with platelets from all animal species studied even at the lowest shear stress used, and LDH loss progressively increased with increasing shear stress amplitude (up to 30% at 50 dyn/cm2), and duration both in the cone-plate and Couette viscometer. These findings indicate a fundamental difference in the response of rabbit, dog, and pig platelets (as compared to that of human platelets) to laminar fluid shear stress in vitro. The mechanism(s) and factors leading to the apparent increased mechanical fragility of the animal platelets as compared to human platelets are currently under investigation.
A NEW FUNCTION OF HUMAN KININOGENS: THE AMINO-TERMINAL REGION OF DOMAIN 1 INVOLVES AN EF HAND-LIKE STRACTURE FOR METAL BINDING
Authors: Higashiyama S , Ohkubo I , Ishiguro H , Sasaki M
Published: August 2018

Two types of kininogens in mammalian plasma, high molecular weight (HMW) and low molecular weight (LMW) kininogens, are the precursors of kinin. Especially, HMW kininogen circulates in the plasma as a complex with prekallikrein and factor XI, and functions as a cofactor in the initial phase reactions of intrinsic blood coagulation cascade. Recently, it has been found that the kininogens have inhibitory activity toward cysteine proteinases. The heavy chain portion, which is identical for HMW and LMW kininogens, is composed of three domains, domain 1, 2 and 3. Each the domain 2 and 3 has a reactive site as a cysteine proteinase inhibitor. However, physiological function of domain 1 remains still unknown. By using the antibody recognizing the interaction between HMW kininogen and Ca2+ (anti-HMW kininogen-Ca2+ antibody) as a probe, we newly found the Ca2+ binding site in the domain 1.

Anti-HMW kininogen-Ca2+ antibody was isolated from anti-HMW kininogen antiserum as an antibody which bound to a HMW kininogen-Sepharose column equilibrated with 40 mM Tris-HCl buffer, pH 7.5, containing 1.0 M NaCl and 1 mM CaCl2, and was eluted with 3 mM EDTA. Resulting from the characterization by ELISA, this antibody specifically recognized the CB-1 region (CNBr-cleavage fragment 1: 1-160 amino acid sequence) of the heavy chain of kininogen molecules in the presence of Ca2+ or Mg2+. Furthermore, circular dichroism (CD) experiments showed that the conformational changes of HMW kininogen and heavy chain were induced by the addition of metal ions such as Ca2+ or Mg2+, and that this change was due to the conformational change of the CB-1 region. The dissociation constant (Kd) for heavy chain measured by Ca2+ titration analysis by CD at 214 nm was found to be 0.33 ± 0.09 mM. The number of Ca2+ binding sites of heavy chain calculated from Hill plot was 1.15 ± 0.04. The EF handlike structure found in the amino-terminal portion of the heavy chain of kininogen molecules strongly supported the above data. This indicates a possibility that kininogens play an important role as a Ca2+ binding protein.
HEPARIN-INDUCED PLATELET AGGREGATION (H-IPA): DOSE/RESPONSE RELATIONSHIPS FOR TWO LOW MOLECULAR WEIGHT (LMW)HEPARIN PREPARATIONS (CY 216 and CY 222)
Authors: Brace L D , Fareed J , Hoppensteadt D
Published: August 2018

We have previously demonstrated that unfractionated heparin causes platelet aggregation (>50%) in about 40% of normal healthy donors tested. H-IPA occurs in a dose-dependent manner and can be inhibited by antogonists of the thromboxane pathway. Using a LMW heparin preparation (PK 10169) and fractions of this agent separated on the basis of molecular weight (MW) by gel permeation chromatography, we showed that H-IPA was dependent upon the MW of the agents tested. In order to further examine this MW dependence, we tested two other LMW heparin preparations, CY 216 (Mol. wt: 5600) and CY 222 (mol. wt: 3800), and 9 subfractions of each of these agents separated on the basis of MW. Blood was drawn from the same donors whose platelets aggregated when heparin was added to their platelet-rich plasma (PRP), and placed into citrate anticoagulant. PRP was prepared, various concentrations of the agents or their fractions were added and aggregation was monitored for 40 minutes at 37°C. Dose/response curves were constructed from the data obtained with each agent. Compared to unmodified heparin with Mr = 15,000 daltons (D), the dose/response curves for CY 216 (Mr = 5000 D) and CY 222 (Mr = 3,500 D) were shifted progressively down and to the right. Dosq' response curves for each of the fractions of CY 216 and CY 222 demonstrated that as the molecular weight of the fractions decreased, the dose/response curves were also shifted progressively down and to the right. These results indicate that as MW decreases, higher concentrations of the fractions are required to cause aggregation, and the maximum aggregation obtained decreases. Fractions with MW less than 2,500 daltons caused aggregation only at concentrations exceeding supra therapeutic range. Since heparin and LMW fractions have inhibitory activity to the activated clotting factors Ila and Xa and LMW fractions have higher anti-Xa than anti-IIa activity, we measured these activities and attempted to correlate them with the ability to cause H-IPA. No correlation between AXa and H-IPA was found. We conclude that the ability to cause H-IPA is an inherent property of heparin and is molecular weight dependent.
IDENTIFICATION OF A SECOND COLLAGEN-BINDING DOMAIN IN HUMAN VON WILLEBRAND FACTOR
Authors: Pareti F I , Nliya K , Kostel P J , McPherson J M , Zimmerman T S , Ruggeri Z M
Published: August 2018

We have recently reported (Journal of Biological Chemistry 261: 15310-15315, 1986) that von Willebrand factor (vWF) possesses a collagen-binding domain localized in a reduced and alkylated tryptic fragment of apparent 52/48 kDa molecular weight extending between residues Val (449) and Lys (728) of the constituent subunit. This proteolytic fragment of vWF also contains a glycoprotein lb-binding domain and a heparin-binding domain. We have now identified a second collagen-binding domain in the Staphylococcus aureus V8 protease-generated fragment I that extends from residue Gly (911) to Glu (1365). The two binding domains exhibit different interaction with collagens of different origin. The reduced and alkylated 52/48 kDa tryptic fragment was a potent inhibitor of vWF binding to equine collagen type I, but had no effect on the binding to bovine collagen type I and III. In contrast, a purified fraction containing the unreduced 52/48 kDa domain inhibited vWF binding to all types of collagen, as did anti-52/48 kDa monoclonal antibodies. Some of these antibodies, however, were more effective in inhibiting binding to equine collagen. On the other hand, fragment I markedly inhibited the binding of vWF to bovine collagen type I and III, but was less effective with equine collagen type I. Direct binding studies using 425j_qabeled fragment I demonstrated that the association constant was 5 to 10 times greater with the bovine collagens than with the equine collagen. The Staphylococcus aureus V8 protease-generated fragment III, which extends from residue Ser (1) to Glu (1365) and contains both collagen-binding domains, was the most potent inhibitor of vWF binding to all types of collagen tested. Thus, vWF has at least two collagen-binding domains. Native conformation appears to be necessary for binding of the 52/48 kDa domain to bovine collagen type I and III, but not to the equine collagen type I tested. The two domains appear to function concurrently in mediating vWF binding to collagen.
EXPRESSION OF HUMAN FIBRINOGEN Aa CHAIN cDNA MUTANTS, TO STUDY THE ACCELERATION OF TISSUE PLASMINOGEN ACTIVATOR (t-PA) INDUCED PLASMIN FORMATION
Authors: Koopman Jaap , Lord Susan T , Haverkate F
Published: August 2018

The capacity to accelerate t-PA induced plasminogen activation, is located on the Aa chain of fibrinogen.

To determine more closely which parts of the Aa chain are involved in the accelerating activity, a number of deletion mutants were constructed using the cloned cDNA. The exact sequence of the mutated cDNA's was determined, using the dideoxy sequencing method according to Sanger et al. Plasmids containing the complete Aa cDNA or the constructed mutants were expressed in E. Coli cells. After treatment with thrombin, the cell lysates showed fibrinopeptide A levels ranging from 40 ng/ml to 200 ng/ml. Western Blotting of the cell lysates, using a monoclonal antibody which reacts specifically with the N-terminal part of the Aa chain of human fibrinogen (Koppert et al., Blood, 66, 503-507, 1985), showed protein bands of the molecular size as predicted by the DNA structure. The complete Aa chain expressed in E. Coli was equally effective in the acceleration of plasmin formation as Aa chain isolated from purified fibrinogen.

Using a number of deletion mutants we showed that the accelerating capacity is associated with amino acids within the Aa stretch 44-199, which is in agreement with previous studies (Nieuwen-huizen, Biochim. Biophys. Acta, 748, 86-92, 1983). This result shows that the approach using molecular biology seems promising for further defining the site(s) involved in the acceleration of t-PA induced plasminogen activation.
FORMATION OF Cl -Cl INHIBITOR AND KALLIKREIN-Cl INHIBITOR COMPLEXES DURING CARDIOPULMONARY BYPASS
Authors: Wachtfogel Yanina T , Harpel Peter C , Henry Edmunds L , Colman Robert W
Published: August 2018

Cardiopulmonary bypass prolongs bleeding time and increases postoperative blood loss. Contact of blood with synthetic surfaces during extracorporeal circulation leads to major qualitative and quantitative alterations in both platelets and neutrophils. Activation of platelets results in thrombocytopenia, decreased sensitivity of platelets to aggregating agents, decreased alpha2-adrenergic and fibrinogen receptors, secretion of thromboxane B2, and depletion of alpha-granule protein contents. Neutrophils,under similar conditions, have also been shown to release their specific granule protein, lactoferrin, and their azurophilic granule enzyme, elastase.We now investigate whether the classical complement, contact, or fibrinolytic pathways have been activated as potential sources of neutrophil agonists. Employing enzyme-linked immunosorbent “sandwich” assays specific for Cl -Cl inhibitor and kalli-krein-Cl inhibitor complexes respectively, we found that plasma levels of both of these formed complexes increased 2fold after clinical cardiopulmonary bypass was completed and reverted to baseline within 24 hours post-operatively. Since these complexes are cleared iji vivo, we investigated their plasma levels during jLn vitro simulated extracorporeal circulation. Over a period of 2 hours, Cl -Cl inhibitor complexes rose from a baseline of 2 + InM to 21 + 2 nM and kalli-krein-Cl inhibitor complexes rose from 2+1 nM to 25 + 5 nM. However, there was no evidence of either in vivo or vitro plasmin-alpha plasmin inhibitor complex formation. These results indicate that activation of the classical pathway of complement and the contact system in plasma may be associated with neutrophil activation seen during clinical cardiopulmonary bypass.
EVIDENCE FOR SYNERGISM BETWEEN TISSUE-TYPE PLASMINOGEN ACTIVATOR AND SINGLE CHAIN UROKINASE ON IN VITRO PLASMA CLOT LYSIS
Authors: Rappaport R S , Blume M R , Vogel R L , Levner M H , Hung P P
Published: August 2018

There is mounting evidence from animal models and the clinic that combination thrombolytic therapy with tissue-type plasminogen activator (tPA) and single chain urokinase (scuPA) is synergistic. Yet, efforts to demonstrate synergism between these two plasminogen activators in vitro have met with discordant results. Collen et al (Thromb. Haemostasis, 56:35, 1986) reported an absence of synergism between these two agents on clot lysis in an in vitro plasma milieu when they were evaluated at molar ratios of 1:4 (tPA:scuPA and vice versa). Gurewich and Pannell (Thromb. Res., 44:217, 1986), however, reported a synergistic effect on fibrin-specific clot lysis in vitro when the agents were combined in concentrations exceeding molar ratios of 1:4 (tPA:scuPA). Here, we present evidence that synergism between tPA and scuPA may be demonstrated in vitro provided that the molar ratio of tPA to scuPA exceeds 1:4 and that the concentration of clot bound or unbound tPA is minimized. In order to achieve this experimental condition, the standard in vitro plasma clot lysis assay was modified. Human plasma clots were incubated first for a short time in plasma containing varying amounts of tPA. After incubation, the clots were washed thoroughly and reimmersed in plasma alone or in plasma containing varying amounts of scuPA or tPA. Under these conditions, lysis proceeded at a greater rate and to a greater extent when tPA clots were immersed in plasma containing an appropriate amount of scuPA than when they were immersed in plasma alone or in plasma containing appropriate amounts of tPA. Lysis of untreated clots or clots exposed first to scuPA and then to plasma containing varying amounts of scuPA proceeded far less efficiently with a characteristic lag. The enhanced lysis produced by tPA and scuPA obeyed the classical definition of synergy: the same biological effect can be obtained with two drugs together at algebraic fractional combinations of less than 1 (Berenbaum, M.C., Clin. Exp. Immunol., 28:1-18, 1977). Thus, conditions that more closely mimic the in vivo situation resulting from a bolus injection of tPA followed by infusion with scuPA, may provide a system for duplication of in vivo synergism in. vi tro and investigation of the mechanism thereof.
RISTOCETIN-INDUCED ENDOTHELIAL CELL BINDING OF PLASMA VON WILLEBRAND FACTOR
Authors: Vokac KA , Ferrars J , Montgomery RR
Published: August 2018

Our laboratory previously used a technique of labeling plasma von Willebrand factor (vWf) with radiolabeled AVW1 - a “ neutral” monoclonal antibody to vWf. This technique has been used to study the binding of plasma vWf to platelets in the plasma milieu. Studies by several laboratories including ours have demonstrated structural glycoproteins on endothelial cells that are analogous to platelet GP Ilb/IIIa and we have shown that the platelet alloantigen Pl-Al is expressed on the surface of cultured endothelial cells. We undertook this study to evaluate the binding of plasma vWf to cultured endothelial cells in confluent monolayer cultures using the “ neutral” monoclonal antibody technique. Plasma vWf was “ labeled” using trace quantities of radiolabeled AVW-1. We then added 80,000 cpm of monoclonal-labeled plasma to 48 well culture plates containing confluent secondary cultures of human umbilical vein endothelial cells. Following the addition of ristocetin, the plates were incubated for 1 hour at room temperature, centrifuged, and the count8 bound and the counts remaining in the supernate were determined. In the presence of ristocetin, 67.5% of the labeled vWf bound to the endothelial cells. When “ labeled“ severe von Willebrand plasma was used or when ristocetin was omitted, less than 5% of the counts bound. Controls using mouse serum or excess mouse IgG to rule out Fc receptor binding and controls to evaluate binding to the subcellular matrix were performed and demonstrated this binding to be vWf and cell surface dependent. Unlike platelet vWf binding, this binding was not inhibited by monoclonal or polyclonal antibodies to platelet GPIb. We studied plasma from patients with type I, a variant of type I, and type Ila vWd and found normal binding with the type I plasma, but reduced binding with the type I variant plasma (14.5%) and the type Ila plasma (7.1%). AVW3, a monoclonal antibody to vWf that blocks vWf binding to platelet GPIb, blocked vWf binding to endothelial cells. Endothelial cells, like platelets, have the ability to bind plasma von Willebrand factor in the presence of ristocetin. This phenomenon occurs on the surface of endothelial cells in culture. Qualitative and quantitative reductions of this vWf binding are found with the plasma of patients with von Willebrand’s disease.
AUTOLOGOUS ENDOTHELIAL SEEDING OF DACRON AORTIC BIFURCATION GRAFTS RESULTS IN DECREASED PLATELET ACCUMULATION
Authors: Örtenwall P , Wadenvik H , Kutti J , Risberg B
Published: August 2018

Autologous endothelial seeding (AES) of vascular prostheses has been found to reduce thrombogenicity in dogs. In the present study, we evaluated the capability of AES to reduce platelet deposition on vascular prostheses in humans.

One limb of an aortic Dacron bifurcation graft was seeded with autologous endothelial cells (EC), harvested from the distal portion of the saphenous vein by enzymatic treatment. The other graft limb was sham-seeded with culture medium, and was used as control. The deposition of Indium-111 labeled platelets was studied 1, 4 and 12 months after surgery. In 17 out of 20 patients, AES resulted in decreased accumulation of radiolabeled platelets, compared to control limbs (p < 0.002), when studied 1 month after surgery. A general reduction in platelet accumulation on vascular prostheses occurred during the first year after surgery. During this observation period a significant difference persisted (p < 0.01), in favor of seeded graft limbs.

Although the seeding density was low (400-5000 EC/cm2), the observed reduction in platelet deposition for AES-treated graft limbs merits further investigation of this technique in humans.
LOCALIZATION OF FUNCTIONAL DOMAINS ON HUMAN PLATELET GP Ib-IX COMPLEX BY EPITOPE ANALYSIS WITH MONOCLONAL ANTIBODIES
Authors: Berndt M C , Du X , Beutler L , Booth W J , Castaldi P A
Published: August 2018

There is now considerable evidence that glycoprotein (GP) Ib plays an important functional role in the von Willebrand factor (vWF)-dependent adhesion of platelets to exposed vascular subendothelium and in the a-thrombin activation of platelets, and that GP IX is important for quinine/quinidine drug-dependent antibody platelet recognition. GP Ib (Mr = 170 KD) consists of two disulfide-linked subunits, Iba (Mr = 135 KD) and Ibβ (Mr = 25 KD), and exists as a heterodimer complex with GP IX (Mr = 22 KD). In this study we have used a panel of 10 antiGP Ib-IX complex monoclonal antibodies to define the functional domains on this complex. Immunoprecipitation of trypsin-treated GP Ib-IX complex revealed that the monoclonal antibodies mapped into three distinct groups: FMC 25, AK 1 and SZ 1, epitopes on the membrane-associated fragment (GP IX and an ≃25 KD remnant of the α-chain disulfide linked to the β-chain); AK 3 and WM 23, epitopes on the central macroglycopeptide core (90 KD); AN 51, SZ 2, AK 2, AP 1 and HIP 1, epitopes on peptide tail (45 KD). Crossblocking studies indicated that with the exception of AK 1 and SZ 1, the monoclonal antibodies were directed against distinct epitopes. All five monoclonal antibodies directed against the peptide tail region blocked ristocetin-dependent vWF-platelet interaction whereas the other five monoclonal antibodies were without effect, indicating that the 45 KD peptide tail region at the plasma end of the α-chain of GP Ib contained the vWF binding domain. Similarly, only the three monoclonal antibodies directed against the membrane-associated region interfered with drug-dependent antibody-platelet interaction.

By western blot analysis, α-thrombin bound to the 45 KD peptide tail region. However, only AP 1 interfered significantly with the α-thrombin-dependent aggregation of platelets. This panel of epitope-defined monoclonal antibodies should be of value in further defining the structure-function relationships of this important membrane complex.
REGULATION OF THE ANTICOAGULANT ACTIVITY OF ACTIVATED PROTEIN C BY PROTEIN S AND PROTEIN S BINDING PROTEIN
Authors: Walker F J
Published: August 2018

Protein S is a vitamin K-dependent protein that acts as a cofactor for the anticoagulant activity of activated protein C both in the proteolytic inactivation of factor V and VIII. Protein S is a single chain protein with a molecular weight of approximately 62 kDa. When the molecular weight of protein S in plasma was determined it was found to be much larger than the single chain protein. The molecular weight of functional protein S when measured by sedimentation equilibrium with the air-driven ultracentrifuge was observed to be between 115 and 130 kDa. In high salt or in the presence of copper ions this was observed to be reduced to approximately 62 kDa. Frontal analysis of plasma indicated that the functional protein by exist in as many as three molecular weight foras. Gel filtration of radiolabeled protein S also indicates heterogeneity in the molecular weight. In order to isolate the binding protein, bovine plasma was fractionated first on a column of immobilized iminodiacetic acid that had been equilibrated with copper ions. The proteins that eluted in the 0.6 M NaCl wash were passed over a column of protein S immobilized on agarose beads. A single protein was observed to elute from the protein S agarose at high salt. Fractionation of human plasma indicated the presence of several proteins. One major component isolated was C4-binding protein. A second major component has also been isolated that appears to correspond to protein S-binding protein that has been isolated from bovine plasma. When added to plasma depleted of both protein S and the binding protein, the binding protein was observed to enhance the anticoagulant activity of activated protein C only in the presence of protein S. Protein S-binding protein was also observed to enhance the rate of factor Va inactivation by activated protein C and protein S.
BLEEDING COMPLICATIONS DURING RT-PA THROMBOLYSIS RELATED TO USE OF ANTI-INFLAMMATORY DRUGS PRIOR TO ACUTE MYOCARDIAL INFARCTION ADMISSION
Authors: Roth A , Barbash G I , Miller H I , Keren G , Laniado S , Seligsohn U
Published: August 2018

Of 57 patients with acute myocardial infarction (AMI) treated with rt-PA, we observed 2 major bleeding complications, both in patients who had been treated with anti-inflammatory drugs prior to admission. The thrombolytic protocol included: lOmg rt-PA in bolus and continuous infusion of 110 mg over 6 hr 5,000 iu heparin in bolus and continuous infusion of 25,000 iu/ 24hr, and aspirin 250 mg/24hr. The first patient, a 64 year old woman had been taking indomethacin 25 mg × 3 daily, during 3 weeks prior to the AMI. Rt-PA protocol was initiated with relief of chest pain and disappearance of ST elevation, but at 2 hr rt, sciatic pain developed. Treatment was continued accor ding to protocol in spite of the pain, but on the 3rd day hemoglobin decreased to 7.8%. Abdominal CT scan disclosed retroperitoneal hemorrhage. All anticoagulant medications were stopped, and 4 units of blood were transfused. The retro-peritoneal mass dissolved gradually. The second patient, a 68 year old male was treated by diclofenac 100 mg for 5 days prior to admission for AMI, and consequently aspirin was removed from the rt-PA protocol. However, 2 hr after completion of the 6 hr rt-PA infusion, gross hematuria and a “coffee ground” vomiting developed. Heparin infusion was discontinued and antacid treatment initiated, resulting in cessation of bleeding within a few hrs. In both patients the anticipated prolongation of APTT (heparin) and about 30% decrease in fibrinogen level were observed as the sole abnormality, and thus we related the bleeding episodes to the anti-aggregating effect of indomethacin and diclofenac respectively. We suggest that the use of anti-inflammatory drugs prior to administration of rt-PA protocol can be hazardous, and that special prudence (possibly protocol modification) is warranted.
PREHOSPITAL CORONARY THROMBOLYSIS: A NEW STRATEGY IN ACUTE MYOCARDIAL INFARCTION
Authors: Teddy Weiss A , Fine David G , Applebaum David , Welber Sima , Sapoznikov Dan , Lotan Chaim , Mosseri Morris , Hasin Yonathan , Gotsman Meryyn S
Published: August 2018

Thirty-four patients with acute myocardial infarction were treated prospectively using a new strategy of pre-hospital intravenous streptokinase given by a physician-operated mobile intensive care unit. Prehospital treated patients who had experienced no previous myocardial infarction were compared to a similar group treated with streptokinase in-hospital. All patients underwent cardiac catheterization on day 6.

Patients receiving streptokinase in the pre-hospital phase of acute myocardial infarction had smaller infarcts and better residual myocardial function than the group given streptokinase in-hospital in terms of peak creatine phosphokinase (900 v.1298 IU, p=0.023), ejection fraction (62 v. 55%, p=0,004), computer-derived dysfunction index (427 v. 727, p=0.003), and electrocardiographic QRS score (4.1 v. 6.4, p=0.001). The only difference between these groups at baseline was the duration of pain prior to initiation of streptokinase therapy (1.0 ± 0.4 hours vs. 1.9 ± 0.9 hours). There were no major complications related to pre-hospital administration of streptokinase.

Pre-hospital stretokinase infusion is feasible, safe and practical. It reduces ischemia time because treatment is not delayed until hospital arrival and therapy limits infarct size. Thrombolytic therapy for acute myocardial infarction can be initiated at home and should not be limited to hospitalized patients.
CARDIAC ENZYME RELEASE AFTER FIBRINOLYTIC THERAPY WITH INTRAVENOUS (i.v.) APSAC OR INTRACORONARY (i.e.) STREPTOKINASE IN ACUTE MYOCARDIAL INFARCTION
Authors: Bonnier J J R M , de Swart J B R M , Hoffmann J J M L
Published: August 2018

The release of CK, CK-MB and ASAT in patients with acute myocardial infarction (AMI) was studied on the relation to reperfusion. In a randomized trial 85 patients with proven AMI entered the study. The occlusion of the infarct related vessel and reperfusion were all assessed angiographically. Fibrinolytic therapy with anisoylated plasminogen streptokinase activator complex (APSAC) i.v. or with streptokinase (SK) i.e. was started within 4 (mean 2.4) hours of the onset of symptoms. 42 were treated with a single i.v. injection over 3-5 min. of APSAC (30U) and 43 with an i.e. infusion over 60 min. of SK (250.000U). Reperfusion was assessed angiographically at 90 min. after the start of treatment. Blood samples were taken before and every 4 hours after dosing up to 24 hours. 74 patients were evaluable for this analysis. The results are:

Conclusion: a statistically significant difference (p < .05)** between the reperfused and non reperfused patients could only be demonstrated for the peak value of CK in the APSAC group and of CK-MB in the SK group.

*On behalf of the Dutch Invasive Reperfusion Study Group.
PROGNOSTIC VALUE OF SPONTANEOUS PLATELET AGGREGATION IN SURVIVORS OF MYOCARDIAL INFARCTION DURING FOUR YEARS FOLLOW UP
Authors: Trip M D , Manger Cats V , Vreeken J
Published: August 2018

Platelet aggregation has been implicated in the pathogenesis of atherosclerosis and its complications. We studied the prognostic value of the presence of spontaneous platelet aggregation (SPA) after myocardial infarction in 165 patients during a four years follow up period. Shortly after infarction 78 (47%) showed SPA and 87 (53%) showed no SPA. There were no differences in sex, age, infarct size or localisation and subsequent treatment between both groups. Patients in the SPA-positive group remained predominantely positive and patients in de SPA-negative group negative during the entire follow up period.

In the SPA-positive group 25(32%) cardiac events (12 × cardiac death, 13× non fatal recurrent infarction) occurred.

In the SPA-negative group 13(15%) cardiac events ( 5× cardiac death, 8× non fatal recurrent infarction) occurred (p < 0.01)

In conclusion: the presence of spontaneous platelet aggregation after myocardial infarction is associated with a higher risk for fatal or non fatal recurrent myocardial infarction.
ACTIVATION OF FIBRINOLYSIS BY PHYSICAL EXERCISE IN HEALTHY INDIVIDUALS WITH DIFFERENT SPORTING ACTIVITIES AND IN PATIENTS AFTER MYOCARDIAL INFARCTION
Authors: Speiser W , Langer W , Pschaick A , Selmayr E , Ibe B , Nowacki P E
Published: August 2018

Regular vigorous sporting activities are known to stimulate blood fibrinolytic capacity estimated by the venous occlution test, and have been reported to have a prophylactic effect against the development of coronary artery disease and myocardial infarction. In the present study, the influence of regular physical exercise on fibrinolysis was studied in healthy individuals and in patients after myocardial infarction taking part in a rehabilitation sports program. The activation of fibrinolysis after bicycle ergometry was investigated in 4 groups of subjects. Group A: 18 healthy male competitive athlets (23 ± 3.5 years of age, mean ± S.D.), Group B: 18
healthy male volunteers (25.7 ± 2.7 years) not engaged in any sporting activities, Group C: 17 healthy male volunteers (50.5 ± 7.7 years) regularly practicing sports, and Group D: 18 male patients after myocardial infarction (54.2 ± 7.9 years). Group A (13.9 ± 2.6 AU/ml, mean ± S.D.) and Group C (15.2 ± 2.9 AU/ml) revealed lower (p-4. 0.05) plasma plasminogen activator inhibitor capacities (PAI-cap) than Group B (18.5 ± 5.5 AU/ml) and Ml-patients (20.7 ± 5.5 AU/ml). All groups showed a significant increase in plasma tissue plasminogen activator (tPA) antigen levels during exercise (Group A: 8.4 ± 6.7 ng/ml; Group B: 7.1 ± 6.2 ng/ml; Group C: 4.9 ± 3.0 ng/ml; and Group D: 3.0 ± 2.9 nl). After exercise significantly higher tPA activities were measured in persons with low PAI-cap at rest (Group A: 5.5 ± 6.4 IU/ml; Group C: 2.9 ± 3.3 IU/ml), whereas Group B (1.1 ± 3.0 IU/ml) and Group D (0.2 ± 0.7 IU/ml) showed only weak activities. Fibrinogen and fibrin split products (D-dimer) levels did not change during exercise testing. The present study indicates that 1. regular vigorous physical exercise enhances blood fibrinolytic activity by reducing blood PAI-cap in healthy individuals, 2. rehabilitation sport is not capable of reducing blood PAI-cap in Ml-patients, and 3. the increased fibrinolytic capacity after physical exercise does not induce systemic fibrinolytic effects.
BLOOD TAURINE AFTER MYOCARDIAL INFARCTION
Authors: Sills T H , Heptinstall S
Published: August 2018

Taurine, an amino acid that is present in high concentrations in the heart, is released from the heart after myocardial damage. There is evidence that the concentration of taurine in whole blood is raised after myocardial infarction (MI), and it has been suggested that blood taurine may be a measure of the degree of infarction. We have obtained serial measurements of blood taurine in patients admitted to a coronary care unit and have compared the results with those obtained for two cardiac enzymes (AST and HBD) and other blood parameters.

The patients were divided into two groups: those for whom there was a peak of AST activity (> 40 i.u./l) (Group 1, n = 24) and those for whom AST and HBD was not raised (Group 2, n = 15). For Group 1 patients, mean results were obtained for each of the parameters for the day on which AST peaked (designated Day 0) and for preceeding and subsequent days. For Group 2 a single mean was obtained. Results marked * in the table differ significantly (p < 0.05 or lower) from those for Group 2:

It can be seen that blood taurine was significantly raised after MI and followed a pattern similar to the neutrophil count. Furthermore, several positive correlations (r = 0.63-0.79) were obtained between taurine and neutrophil count in both groups, but not between taurine and AST or HBD.

In another investigation we measured the amounts of taurine in neutrophils, platelets and plasma from patients with MI (n = 5) and controls (n = 9). We found no differences in the amounts present per neutrophil, per platelet or per ml of plasma.

Our data suggest that the increased level of taurine in blood after MI merely reflects the increased number of neutrophils present in blood following the event.
FIBRINOGEN HETEROGENEITY AND FIBRINOLYTIC ACTIVITY IN AN6I0GRAPHICALLY ASSESSED CORONARY ARTERY DISEASE (CAD)
Authors: Lipinska I , Perlgrund C , Wharton T , Gurewich Y
Published: August 2018

Human plasma was shown previously to contain three forms of thrombin clottable protein: high molecular (HMW) and two lower molecular weight fibrinogen (LMW and LMW*) , the percentage of which in healthy subject is 69, 28 and 32 respectively. Significant abnormalities in the proportion of fibrinogen (Fgb) fractions were observed post–surgically, in cancer, and in patients with myocardial infarction and liver diseases. The aim of this study was to determine Fbg fractions and fibrinolytic activity (FA) in patients with and without proven coronary occlusion. Group I consisted of 21 symptomatic patients without significant coronary obstruction. Group II comprised 116 patients (94 men and 43 women; mean age 65 and 59 y. respectively) who were shown by angiographical examination to have coronary artery occlusions. Plasma Fbg fractions were determined by 3.52 SDS–PA6E of washed fibrin nonstabilized clots dissolved in 8M urea. Quantitation of the fractions were perfomed by densitometric scanning and expressed as percent of total. Fibrinolytic activity was measured in euglobulin fraction using fibrin plate technique. Mean values for HMW, LMW and LMW’ fibrinogen fractions were 64.4±4, 28.8±2 and 6.6±1 for Group I, and 62.5±5, 29.8±3 and 7.7± 1 for Group II respectively. Fibrinolytic activity in Group I and II was 66±26 and 39±24mm2 respectively as compared to 86±35 in healthy subjects. The statistically significant differences between Group I and II were observed for HMW and LMW fractions (p<0.03) and for FA (p<0.001). These results indicate that measurement of blood FA can be useful in the identification of individuals at risk for coronary artery disease. It is also concluded that degradation of plasma fibrinogen is not related to the fibrinolytic activty of blood, but is caused by some other mechanism, as yet not known.
Thrombolytic Properties of Pro-Urokinase, Plasminogen Proactivator (PPA)
Authors: Iga Y , Tanaka K , Tsukada M , Kameyama S , Morichi S , Suyama T
Published: August 2018

Thrombolytic properties of highly purified plasminogen proactivator (PPA) isolated from culture medium of human kidney cells were compared with those of human urinary urokinase(u-UK). When 125-I-PPA or u-UK was added to whole blood perfusion medium containing preformed thrombi made from whole blood by Chandler loop method, the rates of uptake by the thrombi of PPA and u-UK for 4 hrs were only 2.5% and 2.7% respectively. Using the same thrombi and perfusion medium, an in vitro thrombolytic effect of PPA was examined over a 4 hr period. In early perfusion time, a lag phase was observed in the lysis-time curve of PPA, but not of u-UK. After the lag phase, the lysis by PPA increased linearly without early reaching a low level plateau that was characteristic of the lysis by u-UK. At 4hr, the PPA percent lysis was higher than the u-UK figure. Half lives in rabbit of 125-I-labelled PPA and u-UK iv doses were 5.1±0.2 min and 7.3±0.2 min respectively.

The thrombolytic ability of PPA in vivo was evaluated in rabbit pulmonary embolism induced by iv 125-I-fibrin suspension as well as cynomolgus monkey femoral vein thrombosis produced by formation of 125-I-fibrinogen labelled clot in an isolated segment of the vein. In both models, PPA was as effective in thrombolysis as u-UK. However conversion to active UK form, consumptions of plsminogen and PI, fibrinogenolysis and prolongation of APTT in plasma were minor in the PPA group, but not in the u-UK group. It is thus suggested that PPA may cause a local activation limited on the fibrin to lead to a lysis of the thrombi without incurring systemic fibrinolysis
COMPARISON OF A BOLUS AND A INFUSION DOSE REGIMEN OF RECOMBINANT TISSUE TYPE PLASMINOGEN ACTIVATOR (rt-PA) ON THE RATE OF THROMBOLYSIS IN THE RABBIT JUGULAR VEIN MODEL (RJVM)
Authors: Refino C J , Hultgren B , Hollenbach S , Hotchkiss A
Published: August 2018

The success of intravenous infusion of rt-PA in the treatment of myocardial infarction, along with new applications in deep vein thrombosis and pulmonary embolism has stimulated interest in optimizing dosage regimens. Dosage strategies should be tested in a relevant animal model before clinical use. In the RJVM, net lysis is calculated frcm the difference in 1-125 fibrinogen present in a rabbit jugular vein thrombus, pre and post thrombolytic therapy, therefore changes in the rate of lysis would not be detected. To continuously monitor rates of lysis in the RJVM, a flat surface garrma detector probe was secured over the jugular vein thrombus of the anesthetized rabbit, and Gamma counts were accumulated for one minute, at 10 minute intervals. Rates of lysis and net lysis were calculated frcm these data. Net lysis was also determined by the traditional method in order to validate the new technique. Using this experimental design we compared the efficacy of a bolus (0.4 mg/kg over 1 min) to an infusion (0.4 mg/kg over 2 hours, 10% given as an initial bolus). A control group received an infusion of rt-PA excipient. The bolus group was terminated at 60 minutes, since there was no detectable lysis in any of these animals after 30 mins. There was an excellent correlation between the net lysis calculated either by the probe or the traditinal procedure (Y = 1.0 × −2.8, r = 0.88). The lysis rates were:

From these data we conclude that in the RJVM a bolus of rt-PA offers no clear advantage in efficacy over a comparable dose given as a 2 hr infusion. This observation appears to hold true even during the first 30 minutes of treatment where any advantage the bolus might have would most likely be evident
COAGULATION CHANGES UNDER THREE DIFFERENT DOSAGE REGIMENS OF SINGLE-CHAIN UROKINASE TYPE PLASMINOGEN ACTIVATOR
Authors: Zimmermann R , Horn A , Bode C , Harenberg J , Schuler G , Schwarz F , Tillmanns H , Kübler W
Published: August 2018

Therapy with streptokinase and urokinase induce effective thrombolysis but may be complicated by hemorrhagic side effects. In order to minimize these complications single-chain urokinase-type plasminogen activator (scu-PA) was given in patients with acute transmural myocardial infarction at three different dosage regimens consecutively in combination with heparin. In a first group four patients received 15 mg, thirteen 48 mg (group II) and five 72 mg (group III) of scu-PA intravenously. In 22 cases detailed clotting analyses could be performed. Results: The coagulation analysis demonstrated a reduction of the fibrinogen concentration by 11 % in group I, 17 % in group II and 44 % in group III. Plasminogen decreased by 9 % in the first, 43 % in the second and 61 % in the third group. The level of∝2-anti-plasmin showed a reduction of 12 % in group I, 48 % in group II and 80 % in group III. Fibrin degradation products increased in all groups. The euglobulin clot lysis time was moderately shortened in group I but significantly in the groups II and III. Reptilase and thrombin coagulase time were prolonged moderately. Thrombin clotting time and aPTT could be attained at therapeutic range by additional application of heparin. Antithrombin III showed no alteration. Thrombolysis could be achieved in none of the patients in group I but with good results in group II and III.

Our data suggest that intravenous application of scu-PA at a dosage of 48 mg is able to induce effective thrombolysis with only slight destruction of fibrinogen. At higher dosages a further increased rate of thrombolysis may be possible but also a higher rate of fibrinogenolysis may be considered.
THROMBOXANE AND PROSTACYCLIN GENERATION IN CHILDREN UNDERGOING CARDIAC BYPASS SURGERY
Authors: Mclaren M , Shiach C , Gibson B , Pollock J , Lowe G D O , Forbes C D
Published: August 2018

Children undergoing surgery involving cardiac bypass frequently have problems with post-operative bleeding, more so than children having the same length of surgery but without cardiac bypass. Although the platelet count is known to fall during bypass surgery it also falls in otter groups of surgical patients inwhom post-operative bleeding is nota problem. The passage of blood through the bypass machine may cause damage to the platelets which may therefore be functionally abnormal after surgery and thus promote bleeding. We studied eight patients undergoing cardiac bypass surgery aged between 4 and 14 years.All had similar operating conditions and non-pulsatile , membrane oxygenatory bypass. Each patient was sampled immediately prior to surgery after being anaesthetised and 30 minutes and 24 hours post-operatively. Platelet count, anti thrombin III and proteinC levels fell significantly consistent with activation of platelets and coagulation. Plasma levels of beta-thromboglobulin, thromboxane B2 and prostacyclin metabolites (all measured by radioimmunoassay) were elevated in most patients 30 minutes after surgery, but had usually returned to normal levels 24 hours later. We conclude that cardiac bypass in children causes transient activation of platelets and the thromboxane/prostacyclin pathways: the relationship to bleeding requires further study.
FRAGMIN VS HEPARIN AT RECYCLING OE HUMAN BLOOD IN HEART-LUNG MACHINE (HLM)
Authors: Bagge L , Holmer E , Nystroöm S O , Tydeén H , Wahlberg T
Published: August 2018

During cardio-pulmonary bypass (CPB), Heparin inhibits EXa (EXal), thrombin and platelet activity and is also reported to induce fibrinolysis. Eragmin (Erag) has 25% thrombin inhibition capacity as related to that of Heparin (Hep). An in_vitro study was performed to compare Frag with Hep by circulating blood in a pure artificial system. In 20 experiments, 400 ml of freshly collected blood with Frag or Hep were recycled for 2 h. HLM was primed with 400 ml of Ringeracetate. Blood sampling: donor, blood pack and every 20 min from the oxygenator. V_a£i£ble£/jassay/:ACT/Hemochron/5 APTT , TT and NT/Nyegaard/;FXaI, FVIII and ATIiT t"ATA)/amydolytic/; AT 111 (ATAg) and vWF/IEP/;Plasminogen (Pig) and albumine/immuno-diffusion/;FDP/Wellcome/;Platelet function/Adeplat S/;Fibrinogen (Fbg)/clottable/;Hemolysis (HL)/photometric/; (β -Thromboglobulin ((βTG)/RTA/;EVF, Hb, platelet count (PC) and Leucocyte count (LC)/ conventional). Corrections for hemo-/plasma dilutions were calculated. Dosages (n): Frag: 750 (1), 1500 (3), 2100 (4), 2500 (4) FXal-U (U); Hep: 1000 (3), 1500 (6) IU clinical level. Clotting only occurred at Frag 750 (1) and 1500 (2) U, when ACT, APTT, FVIII, Fbg and ATA were significantly lowered. Generally, PC fell 75% during the recycling, while PF was constant'∼20% and (βTG increased. Neither presence of FDP nor Pig consumption were detected. FXal, ACT, APTT, TT and NT were dose dependent for both drugs. ATA was directly dose-related to Frag but inversely to Hep. LC decreased with the Frag-dose but inversely to that of Hep. HL increased generally. Several proteins increased (clotting excl): Fbg 30%, ATAg 25%, ATA 45?o and vWF 60%. Conclusions. Prevention of clotting required about the double dosage of Frag. Shortened ACT and APTT predicted clotting while the levels of FXal, TT and NT did not. Thus, an effective thrombin inhibition is needed under this conditions. Consumptions of FVIII, Fbg and ATA but no further drop in PC at clotting, indicate weak platelet aggregation involvement. Absence of fibrinolytic signs supports that the fibrinolysis seen at CPB, is not a genuine effect of Hep (or Frag). Increases in some proteins may be caused by cytolysis. The rise in vWF is probably due to release from platelet surfaces.