bluuebunny/crossref_metadata_2025 huggingface dataset:
Overview: Reaction To Two Types Of Thrombus Deposition
Authors: Moore S , Belbeck L W , Richardson M
Published: July 2018

Damage to the luminal surface of an artery is associated with the development of platelet-fibrin thrombus or a monolayer of platelets. The first type of reaction (Type I) follows damage caused by an indwelling catheter or by repeated exposure of the endothelial surface to antibody. The second (Type 2) follows removal of the endothelium by a Fogarty balloon catheter. The reaction of the vessel wall to these two types of injury, while having many similar features, differs in a number of important respects. Both are dependant on the interaction of platelets with the vessel wall and can be suppressed or inhibited by inducing a severe thrombocytopenia using anti-platelet serum. In both, the thickness of lesions is related to repetitiveness of injury. Both are characterized by smooth muscle cell proliferation and lipid accumulation in normolipemic rabbits. The type I lesion regresses rapidly, losing size and lipid content, when the injury stimulus is removed. The Type 2 lesion persists and continues to accumulate lipid over time

In the Type I lesion, induced by an indwelling aortic catheter, platelet consumption is increased for eight days and fresh platelet-fibrin thrombus may be found on the surface of the lesion months after placement of the catheter. Platelet survival is shortened. In the Type 2 lesion the initial interaction of platelets with the vessel wall is short lived, and the surface covering of platelets is rapidly lost. Platelet survival is not shortened in rabbits. While both lesions exhibit abundant accumulation of extracellular and intracellular lipid, this occurs earlier in the Type I lesion and disappears fairly rapidly when the Injury stimulus is removed. Lipid appears later in the Type 2 lesion, persists and increases in amount, even following a single injury. Fatty streaks, though observed in both types of injury, are more likely to be seen as a stage of regression of Type I lesions. The Type I lesion closely resembles the raised (complicated) lesion of human atherosclerosis. The Type 2 lesion resembles the human fibrous plaque. Supported by Grants MT-2168, M.R.C., 15-7 O.H.F.
Platelet-Fibrin Deposition On Subendothelium In Conenital Bleeding Disorders
Authors: Weiss H J , Turitto V T , Baumgartner H R
Published: July 2018

In order to identify components of the blood which are important in hemostasis-thrombosis, and to clarify the hemostatic defect in patients with bleeding disorders, we have measured various parameters of platelet dimensions and fibrin deposition on everted de-endothelialized rabbit aorta in an annular chamber. Studies were done by drawing venous blood directly through the chamber at various shear rates; platelet-fibrin deposition was measured morphometrically. At high shear rates (2600 sec-1 and above) the defective adhesion observed in von Willebrand’s disease (vWD) and the Bernard-Soulier Syndrome (BSS) is due to a requirement of the F.VIII/vWF and GPI complex for the initial contact of platelets with the subendothelium. The defect is enhanced with increasing shear rate. Reduced thrombus stability is also seen in vWD. At a shear rate of 650 sec-1 perfusion of normal blood for 5-10 min results in 65-95% surface coverage with fibrin. Both fibrin deposition and platelet thrombus dimensions were markedly decreased in patients with severe deficiencies of factors VIII and IX (PTT 100-140 sec), in the BSS, and in a patient with deficient platelet binding of Va-Xa. In contrast, normal (or near-normal) values were obtained in patients with severe deficiencies of factors XI and XII (PTT 100-500 sec). Fibrin formation was normal or increased in thrombasthenia. These findings provide a basis for identifying important components of the hemostatic mechanism on physiologic surfaces in flowing blood and may explain why some patients with deficiencies of factor XI and XII do not bleed.
Diagnostic Management Of Pulmonary Embolism (PE)
Authors: Robin Eugene D
Published: July 2018

The diagnostic management of PE should be based on the hazards of a suprious diagnosis versus the hazards of a missed diagnosis. Clinical diagnosis of PE is notoriously unreliable. Ancillary tests such as routine chest x-ray, blood gases, and EKG are non-sensitive and non-specific. Perfusion lung scanning has high sensitivity but low specificity. Thus a negative perfusion scan tends to rule out PE, but a positive scan does not mean PE. Ventilation scans are worthless when the perfusion scan is negative and lead to unacceptable errors when positive. Angiography is the gold standard of diagnosis. When negative, PE is operationally ruled out. When positive, PE is an overwhelming indication for treatment. Approximately 60% of patients who are suspects require angiography for optimal decision making.
Fibrinogen Biosynthesis: In Vitro Translation. Glycosylation And Translocation Of Fibrinogen Peptide Chains
Authors: Fuller G M , Nickerson J M
Published: July 2018

Fibrinogen is a hepatically derived plasma glycoprotein that is composed of three pairs of nonidentical chains linked together by complex sets of disulfide bridges. In an effort to understand the molecular and cellular processes of translating and assembling this important multichained protein we have utilized an in vitro translating system using mRNA’s for rat fibrinogen. Highly specific antibodies to fibrinogen and to each chain have been developed and used to immunoprecipitate the nascent Aα, Bβ, and γ polypeptides. We have also used a rat hepatoma cell line which synthesizes and secretes fibrinogen to prepare nonglycosylated but processed fibrinogen subunits. SDS/PAGE analysis of the translation products clearly show that each polypeptide has a “signal” peptide located at its amino terminal end. The size of the signal peptide is different for each chain. These results demonstrate that separate mRNA’s exist for each of the fibrinogen subunits. Temporal analysis of the glycosylation of the Bβ and γ chain reveal that the γ chain receives its Asn-linked carbohydrate as an early cotranslational event. The Bβ chain’s core carbohydrate moiety is near the end of the polypeptide and our evidence shows that the glycosylation event likely occurs posttranslationally. When microsomal membranes are added to an on-going translation system, all three of fibrinogen's polypeptides translocate into the cisternal space, with an apparent equal stiochiometry. Additional experiments suggest that fibrinogen assembly occurs as a cotranslational process.

These studies have been supported in part by NIH HL - 16445 and HL 00162.
Consumption Of Viiirrag During Delayed Coagulation Process
Authors: Hada M , Ikematsu S , Fujimaki M , Fukutake K
Published: July 2018

Coagulational functions of VIIIR:AG are still unknown, while its serological significance in laboratory medicine has been established. In this study it will be analyzed the mechanism of the consumption of VIIIR:AG during the process of prolonged blood coagulation.

Quantitative assay of VIIIR:AG in plasma and serum is measured by Laurell’s method using 1% agarose with anti-F.VIII rabbit serum and qualitative assay of VIIIR:AG is performed by crossed-immunoelectrophoresis with anti F.VIII rabbit serum.

In 28 patients with Hemophilia A a negative relationship between serum VIIIR:AG / plasma VIIIR:AG ratio and prolongation of PTT is estimated and also a positive relationship between serum VIIIR:AG / plasma VIIIR:AG ratio and serum VIII:vW / plasma VIII:vW ratio is obtained. However, serum VIIIR:AG shows the similar quantity to plasma VIIIR:AG in the cases within normal clotting time, which have been treated by the in vitro addition of thrombin or by transfusion of AHG in vivo. When heparin or synthetic antithrombin ( MCI-9038 ) is added into normal whole blood or F.XIII deficient whole blood, the case of normal whole blood shows delayed clotting time and decrease of serum VIIIR:AG, but the case of F.XIII deficient whole blood indicates no decrease of serum VIIIR:AG. Furthermore, in those conditions change of the concentration of CIg in serum, which has been pointed out with fibrin crosslinking, indicate the similar behavior as serum VIIIR:AG.

The results obtained above might suggest that serum VIIIR:AG tend to decrease in the cases with prolonged clotting time, and the F.XIII activity might be involved in the consumption mechanism of serum VIIIR:AG in such an abnormal condition, as fibrin crosslinkage could not carry out properly.
Separation Of Ahf From Vwf By Solid-Phase Polyelectrolytes (PE); Further Evidence For Their Separate Identity
Authors: Johnson Alan J , Soberano Mercedes E , Kowalski Steven , Fulton Anne , MacDonald Valerie E , Semar Martin
Published: July 2018

The nature of the AHF/vWF complex in plasma, cryoprecipitate, or intermediate purity concentrate has been the subject of intensive study. Some investigators have felt that it was a single molecule with at least two activities; others have thought that it was made up of two noncovalently bound molecules, partially separable by Sepharose chromatography or ultra-centrifugation in high ionic strength NaCl or CaCl2, or by interaction with specific antibodies. Our data indicate that AHF from human, porcine or bovine plasma, human cryoprecipitate, or intermediate purity AHF concentrates may be quantitatively separated from vWF by adsorption of the AHF on solid-phase maleic anhydride copolymer resins; most of the AHF may be eluted from the resin by a high concentration of salt while virtually all the vWF remains in the supernatant; the separation is unaffected by usual concentrations of protease inhibitors (DFP, 1mM) benzamidine (1mM), PMSF (1 mM) or trasylol (10-100 KIU/ml). One adsorption changes the ratio of AHF/vWF units in plasma from 1:1 to approximately 200:1; after two adsorptions the ratio is approximately 1500:1. AHF dissociated from vWF assays the same by one and two stage assays, is neutralizable by specific homologous antibody and is activatable by thrombin. When vWF in cryoprecipitate was separated from AHF by adsorption of the AHF on PE, the ratio of vWF to AHF was 200:1. At concentrations greater than 0.1 units/ml, vWF levels, as assayed by the ristocetin cofactor assay, were comparable to results obtained by the RIA of Hoyer, and the solid-phase radio-immunoassay of Ruggieri. These data are consistent with a non-covalent AHF/vWF complex made up of two different molecules which may be separated readily by adsorption on solid-phase PEs.
Activation Of Hageman Factor By Heparin
Authors: van Rosevelt R F , Armstrong A L , van Mourik J A
Published: July 2018

Exposure of plasma to certain negatively charged substances, leads through the action of activated Hageman factor (HF) to generation of bradykinin (bk), a peptide cleaved from HMW-kininogen by kallikrein. We designed a method which permits rapid measurement of plasma Bk by RIA. This method provides a specific and sensitive means to study contact activation in whole blood and plasma. Heparin (Hep) was found to be effective in the activation of HF even at low concentrations (1,25 U/ml plasma). The rate of activation was dependant on the mol. weight of the Hep studied. Upon incubation of HF deficient plasmas with high concentrations of Hep (20 U/ml), no Bk generation was observed. The capacity of Hep to induce Bk-generation in different normal plasmas varied considerably. Upon incubation of plasma from 8 individuals with Hep (5 U/ml) at 0’C for 2 h, the increase of the Bk concentrations ranged from 20 to 770 ng/ml plasma. Evidence was obtained that “cold promoted activation” (CPA) accounted for these differencies. When CPA-positive plasmas (as judged by shortening oF the thrombotesttime after incubation overnight at 4’C) were incubated with Hep (5 U/ml) for 2 h at 0’C, the Bk-concent rat ions increased from 1 to 375 ± 75 ng/ml (mean ± SEM), wheras in the absence of Hep less than 10 ng Bk/ml/2h was generated. However, when CPAnegative plasmas were incubated with Hep, the increment of Bk was only 25 ± 20 ng/ml 2h. We assume that Hep and the active principle present in CPA positive plasma act synergetically in HF activation. Similarly, the action of dextran sulphate (DS) on HF was potentiated by Hep. When plasma (CPA negative) was incubated with either low concentrations of DS (<1 μg/ml) or Hep (2-5 U/ml), only minor amounts of BK were generated. When Hep and DS were added together to plasma, the amount of Bk generated increased up to 50-fold under these conditions. We conclude that in plasma, heparin is an activator of HF, notably in the presence of other activators of the contact system.
The Kinetics Of The Inhibition Of Human Plasma Kallikrein By Plasma Protease Inhibitors: Role Of High Molecular Weight Kininogen
Authors: Schapira M , James A , Scott C F , Kueppers F , James H L , Cohen A B , Colman R W
Published: July 2018

Plasma kallikrein (KAL) is inhibited by several plasma protease inhibitors, including C1-inhibitor (C1-INH), antithrombin III (ATIII), α1-antitrypsin (α1AT), and α2-macroglobulin (α2M). To assess the mechanism of action and the relative importance of these inhibitors, we have undertaken inhibition studies with purified proteins, using H-D-Pro- Phe-Arg-Nan as KAL substrate. Inhibition was competitive with C1INH, ATIII, and α1AT and noncompetitive with α2M. KAL retained 14% of its catalytic efficiency when complexed to α2M. The rate constants for inhibition by C1INH, ATIII, α1AT, and α2M were 28, 0.18, 0.003, and 6.9 M-ls-1(10-3) respectively. Michaelis-Menten kinetics was observed for the inhibition by ATIII, αlAT, and α2M. The constants for the rate-limiting formation of the irreversible complexes were 16, 0.27 and 2.0 s-1(xl02), while the KI’s for the reversible complex were 86, 63, and 0.29 γM, respectively for ATIII, α1AT and α2M. In_contrast, no Michaelis-Menten complex was observed when C1INH inhibited KAL. These results indicate that (a) C1INH is the most efficient inhibitor of KAL, (b) α2M is a significant inhibitor of KAL, (c) both ATIII and αlAT are probably not significant inhibitors of KAL. We have shown that high molecular weight kininogen (HMWK) decreases the inactivation rate of KAL by C1INH by forming a reversible complex with KAL. We now report that the reaction rates of KAL with ATIII and α1AT, which are competitive inhibitors, were decreased by 50%, when HMWK was 1 U/ml or 0.73 γM. When KAL was inhibited by α2M, a noncompetitive inhibitor, the inactivation rates were identical in the presence or absence of HMWK. Since HMWK protects KAL from being inhibited by competitive inhibitors but not by a noncompetitive one, these results confirm our previous observation indicating that the binding site for IMWK on KAL is closely linked to its catalytic site.
Kallikrein-Like Activity Of Crotalase, A Snake Venom Enzyme Which Clots Fibrinogen
Authors: Markland F S , Shaw E , Kettner C , Schiffman S , Bajwa S S , Reddy K N N , Kirakossian H , Pirkle H
Published: July 2018

During amino acid sequence determination of crotalase, a thrombin-like enzyme from Crotalus adamanteus (eastern diamondback rattlesnake) venom, we found that in addition to the expected structural homology with bovine thrombin, there was even greater homology with porcine pancreatic kallikrein. In exploring further the similarity between crotalase and kallikrein, several completely unexpected and interesting observations were made. First, crotalase was rapidly and specifically inhibited by the tripeptide, affinity-labelling chloromethyl ketone inhibitor Pro-Phe-Arg-CH2Cl, which is known to be a specific inhibitor of urinary kallikrein. Further, crotalase exhibits significant activity not only with the thrombin chromogenic substrate S-2238 (H-D-Phe- pipecolyl-Arg-p-nitroanilide) and the general serine protease substrate S-2160 (N-benzoyl-Fhe-Val-Arg-p-nitroanilide) but also with the plasma kallikrein substrate S-2302 (H-D- Pro-Phe-Arg-p-nitroanilide) and the glandular kallikrein substrate S-2266 (H-D-Val-Leu-Arg-p-nitroanilide). Additionally, SDS polyacrylamide gel electrophoresis reveals that crotalase cleaves the plasma kallikrein susceptible bonds in human high molecular weight kiniogen (HMWK) producing intermediates with procoagulant activity. Analyses for bradykinin release from HMWK are presently in progress. Interestingly, one of the normal activities of plasma kallikrein, the activation of human plasminogen, was not one of the activities possessed by crotalase.

In summary, it would appear that crotalase has significant kallikrein-like activity. Whether this will prove to be of importance in the ongoing clinical application of fibrinogen clotting snake venom enzymes, such as ancrod and batroxobin, remains to be shown.
Monitoring Of Human Kidney Transplants Using Quantification Of Autologous 111Indium -Oxine Platelet Label Deposition - Beneficial Effect Of PGI2-Treatment In Acute (AR) And Chronic Rejection (CR)
Authors: Sinzinger H , Leithner Ch , Schwarz M
Published: July 2018

Eighty patients with AR and chronic transplant rejection as well as with a stable transplant function were monitored by a combination of quantification of 111In-oxine labeled platelet uptake in the transplanted kidney (under γ- camera) and platelet survival (PS). In acute phase after transplantation the patients were controlled twice or three times a day until 3-4 weeks (weekly platelet labeling) after the transplantation. The quantification of platelet deposition (index (i): transplant region:contralateral region/cts. per 5 minutes) is demonstrated to be a very useful parameter of early diagnosis of rejection crisis. In AR i reaches values up to 2,00, the PS between 24 and 72 hours. In constant transplant function i below 1,20 are found, indicating that also in patients without any clinical sign of rejection, a platelet deposition and a shortening of PS occurs. In 8 patients with CR (i: 1,201,35; PS: 60-96 hours) and one week continuous i.v. PG2-administration (5ng PG2/kg/min) leads to a decrease in platelet deposition (i: minus 0,10-0,15) and a prolongation of PS. In 4 of these patients also the serum creatinine was decreased between 0,5 and 1,1 mg%. Using thereafter a combination of antiplatelet drugs we try now to retain the platelet deposition at the lower level reached by means of the PGI2application. Similar beneficial data could be obtained by treating acute rejection with PGI2-infusion. The preliminary data of a controlled study and the platelet function behaviour during PGI2 are reported.
Cardiopulmonary Dysfunction And Changes In Production Of TXA2 And PGI2 Induced By Intracoronary Injection With Arachidonic Acid
Authors: Isohisa I , Tanoue K , Ariga S , Sakakibara C , Yamazaki H
Published: July 2018

A 4 Wedge-Pressure catheter was inserted to 1 cm above aortic valve in ascending aorta through right common carotid artery of 16 rabbits. 1.5 mg/kg of sodium arachidonate (AA) was injected into the proximal portion of ascending aorta after inflation of balloon which obstructed the ascending aorta. Inflation remained for 5 sec, the solution was perfused into coronary arteries. ECG, respiratory movement and blood pressure were recorded. Before, 3 and 60 min after AA, 10 ml of heparinized blood was collected, added with 0.1 ml of Tris-HCl buffer (pH 8.0) containing indomethacin (10 μM in final concentration) and centrifuged to collect PPP for measurement of TXB2 and 6-Keto PGF1α using radio immunoassay. 8 rabbits were injected with 10 mg/kg of (E)-3-[4-(3- pyridylmethyl)phenyl]-2-methyl-2-propenoic acid (OKY-1580, a specific inhibitor on TXA2 synthetase) 3 min before AA injection. In control, arrhythmia and marked ST depression appeared in all cases after AA. Apnea was seen in 4 and 1 case died 7 min after AA. In OKY group, arrhythmia and apnea were not seen. 2 cases showed ST depression. Blood pressure decreased in both the groups similarly. TXB2 values were 1.091+0.498 ng/ml (mean+SD) before, 3.037±0.927 3 min after and 1.014±0.426 60 min after in control. In OKY group those were 1.3154±). 926, 0.830±0.334 and 0.764±0.343. There was a significant difference in the values 3 min after between both the groups. In control, 6-Keto PGF1α values were 3.337± 2.279 ng/ml before, 26.668±16.353 3 min after and 3.975± 3.548 60 min after. In OKY group, those were 2.151±0.528, 61.064±21.420 and 4.264±2.530. There was also a significant difference in the values 3 min after. Histologic findings of heart showed more remarkable ischemic changes in control than in OKY group. These findings suggest that the intracoronary injection with AA induced ischemic changes in heart through TXA2 synthesis at least partially and the changes were able to be prevented by OKY 1580.
Plasma β-Thromboglobulin, Platelet Factor 4 And Fibrinopeptide A In Patients With Coronary Artery Disease
Authors: Nichols A , Owen J , Kaplan K L , Cannon P J , Nossel H L , Sciacca R
Published: July 2018

To determine whether activation of platelets and coagulation is present in patients with coronary artery disease, plasma levels of platelet factor 4 (PF4), β-thromboglobulin (βTG), and fibrinopeptide A (FPA) were measured by radioimmunoassay in patients subjected to coronary angiography. The patients were divided into those with normal coronary angiograms (Group I, n = 14), those with coronary artery disease (> 70% narrowing) but no previous myocardial infarction (Group II, n = 32), and those with coronary artery disease and documented previous myocardial infarction (Group III, n = 36). The three groups did not differ in sex, incidence of hypertension or diabetes, serum cholesterol, HDL cholesterol, BUN or platelet count. Geometric mean values for the three groups were FPA: 0.77, 0.81 and 1.01 pmol/ml respectively, βTG: 22.7, 21.6 and 33.2 ng/ml respectively, and PF4: 5.7, 5.8 and 8.3 ng/ml respectively. When the data were tested by analysis of variance, significant elevations of βTG (p< .01) and PF4 (p< .05) were found in Group III but there were no other significant changes. When Group III was subdivided into patients with and without ventricular aneurysm, BTG and FPA levels were found to be higher in patients with aneurysm than without: βTG 45.9 vs. 30.3 ng/ml and FPA 1.64 vs. 0.88 pmol/ml (p< .05 for each). βTG levels were also higher in patients with congestive heart failure (p< .01) and showed an inverse correlation with left ventricular ejection fraction (p< .05) and a direct correlation with the extent of left ventricular asynergy (p< .01). In conclusion, elevations in βTG and PF4 were associated with previous infarction, not with coronary artery disease. These changes are thought to reflect platelet reaction with the damaged ventricular wall. Elevations in FPA were seen only in patients with ventricular aneurysm and may reflect mural thrombus within the aneurysm.
UK-37,248, A Novel, Selective Thromboxane Synthetase Inhibitor With Anti-Aggregatory And Anti-Thrombotic Activity
Authors: Randall M J , Parry M J , Hawkeswood E E , Cross P E , Dickinson R P
Published: July 2018

UK-37,248, 4-(2-(lH-imidazol-l-yl)ethoxy)benzoic acid hydrochloride, potently inhibited human blood platelet microsomal thronfooxane (Tx) synthetase, IC50=3xl0-9M. TxB2 was quantitated by a specific radioimmunoassay (RIA). In contrast (PG) endoperoxide synthesis by ram seminal vesicle microsomes and prostacylclin (PGI2) synthesis by pig aortic microsomes/were minimally affected by concentrations of UK-37,248 up to 1x10-4M. In the rabbit isolated perfused lung arachidonic acid (AA) metabolites were quantitated by differential bioassay. Concentrations of UK-37,248 from 10-7-10-6M selectively reduced TxA2 production from AA but increased the release of PGI2 and other PGs.

The effect of UK-37,248 in the whole animal was studied by estimating TxB2 levels in blood samples removed before and after dosing. The samples were allowed to clot and the serum TxB2 levels were assessed by RIA. In anaesthetised rabbits, 15 minutes after injection of 0.3mg/kg i.v. UK-37,248, serum TxB2 levels were reduced by 75%. In dogs the compound was similarly effective, lmg/kg p.o. inhibiting TxB2 production by 79% two hours after dosing. Aggregation of human platelet-rich plasma in vitro, initiated by threshold collagen, was inhibited by UK-37,248 (IC50=4.8x10-6M). In rabbits, UK-37,248 at 2mg/kg i.v. prevented the mortality due to pulmonary embolism and reduced the associated thrombocytopenia and elevation of plasma TxB2 caused by i.v. AA. In conclusion, UK-37,248 is a selective inhibitor of platelet Tx-synthetase with antiaggregatory and anti-thrombotic activity.
Effect Of An Inhibitor Of Thromboxane Synthesis (UK-37, 248-01) On Thrombin Induced Pulmonary Insufficiency In The Rat
Authors: Sandler H , Saldeen T
Published: July 2018

Severe trauma in man may be followed by an acute and/or delayed respiratory insufficiency due to microembolism in the lungs, the early and delayed microembolism syndrome, respectively. These conditions can be simulated in the rat by injection of thrombin and a fibrinolysis inhibitor. We have investigated the effect of a new inhibitor of thromboxane synthesis in this model.

Intravenous injection of 500 IE thrombin/kg body weight in rats pretreated with trans-4-aminomethyl-cyclohexane-carboxylic acid 200 mg/kg b.w. resulted in a rapid, strong increase in the pulmonary arterial pressure (PAP) and a decrease in the mean arterial pressure (MAP). These reactions were followed by a progressive, slow increase in PAP, a slow decrease MAP and a heavy increase in the weight of the lungs due to interstitial and alveolar oedema.

In rats pretreated with UK-37, 248-01 (Pfizer, England) 4 mg/kg b.w. the immediate increase in PAP and decrease in MAP was strongly inhibited. No effect on the later changes in PAP, MAP or lung weight was seen.

The results speak for the release of thromboxane A2 being of importance in the early microembolism syndrome but not in the delayed syndrome. Thromboxane may be synthesized in platelets or leucocytes trapped in the lungs. Rats with depletion of the polymorphonuclear cells due to injection of a leucocyte antiserum reacted to the thrombin injection in the same way as rats pretreated with UK-37, 248-01 indicating a relationship between thromboxane A2 and the leucocytes trapped in the lungs.
Measurement Of In Vivo Fibrinolysis: A Comparison Of Assays For Fibrinogen Fragments E And Bβl-42
Authors: Lane D A , Knight I , Ireland H , Mitchell T R
Published: July 2018

The optimal method for measuring in vivo fibrinolysis has yet to be defined. In the present study we have compared methods that measure two different fragments produced when circulating plasmin digests fibrin(ogen). Double antibody radioimmunoassays were set up to measure fibrinogen fragments E (FgE) and Bβ1-42 and these detected less than 0.04 to 0.02 pmol/ml, respectively. Blood was collected into an anticoagulant containing EDTA, heparin, aprotinin and e aminocaproic acid and fibrinogen was removed with Reptilase or by ethanol precipitation. Normal plasma concentrations (n = 20) of FgE and Bβ1-42 were found to be 3.10 and 1.59 pmol/ml, respectively. Patients with heart valve replacements (n = 6) had mean FgE and Bβ1-42 plasma concentrations of 5.07 and 3.06 pmol/ml, respectively. Patients recovering from acute thrombotic stroke (n = 24) had elevated mean FgE and Bβ1-42 levels, 6.52 and 3.80 pmol/ml respectively, but the correlation between the individual results was poor. Sequential blood samples were taken from patients (n = 15) undergoing hip replacement operations. Pre operative mean plasma concentrations were 5.82 and 3.40 pmol/ml, respectively. Peak mean FgE plasma concentrations occurred on post operative day 1, 9.03 pmol/ml, while the Bβ1-42 maximum was on post operative day 4, 8.0 pmol/ml. Patients (n = 6) with renal failure had mean FgE and Bβ1-42 plasma concentrations of 5.39 and 36.9 pmol/ml, respectively. Haemodialysis increased these levels to 8.18 and 60.0 pmol/ ml, respectively, but not all of the patients exhibited an increase. It is concluded that (a) assays for FgE and Bβ31- 42 measure different aspects or stages of the vivo fibrinolytic process (b) following renal failure there is either a large production of early plasmin degradation fragments, or more likely, Bβ1-42 is inadequately catabolised by the kidney.
The Active Site Of Antithrombin. Release Of The Same Proteolytically Cleaved Form Of The Inhibitor From Complexes With Factor IXa, Factor Xa And Thrombin
Authors: Björk I , Jackson C M , Jörnvail H , Lavine K K , Nordling K , Salsgiver W J
Published: July 2018

Reactions between near-equimolar amounts of antithrombin and Factors IXa or Xa resulted in the formation of a free, proteolytically modified, two-chain form of the inhibitor, in addition to the inactive anti thrombin-protease complexes. The modified antithrombin produced by either enzyme behaved electrophoretically identical to the previously characterized modified inhibitor formed in the reaction with thrombin. Like in the latter reaction, the formation of the modified antithrombin by Factor Xa was increased in the presence of heparin, while only small amounts were produced by Factor IXa both in the absence and presence of the polysaccharide. Amino-terminal sequence analyses of the isolated modified inhibitor formed by Factor Xa showed that a single Arg-Ser bond in the carboxy-terminal end of the inhibitor had been cleaved. This cleavage site is identical to that previously identified in free thrombin-modified antithrombin. The antithrombin-Factor IXa and antithrombin-Factor Xa complexes were purified and dissociated by ammonia or hydroxyl amine. The dissociation products of both complexes were free enzyme and a modified two-chain form of the inhibitor. Electrophoresis studies and amino-terminal sequence analyses showed that the modified antithrombin obtained from either complex was identical to that produced in free form by the enzymes and also to the modified inhibitor that was shown previously to be released from the antithrombin-thrombin complex. The fact that identical findings were obtained for the reactions between anti thrombin and three enzymes with different specificities strongly supports the previous proposal that the observed cleavage site is the active site of antithrombin.
Differential Uptake Of 111-Indium By Platelets And Granulocytes
Authors: Hawker R J , Hall Carolyn E
Published: July 2018

Mixed cell,leucocyte or platelet preparations labelled with 111-Indium oxine have been used to visualise septic foci, renal rejection and venous and arterial thrombosis. Pure polymorphonuclear leucocyte preparations are difficult to prepare without modification of cellular activity and are generally contaminated with platelets. Up to 27% of the Indium label is associated with platelets when a granulocyte preparation contains as little as 2% of the original platelet population.

Under conditions of limiting Indium-oxine,uptake by platelets is 16.8 and 17.5 times more effective than granulocytes or erythrocytes per unit area of membrane; the accumulation by these latter cell types is related to their surface area with a ratio of 4.7:1. This data is further evidence for a facilitated mechanism for the uptake of Indium by platelets.

Labelled platelets have been used for visualisation of sterile and septic abscesses in animals, and in man minor inflammatory responses can be detected. 99.8% pure platelet preparations are simple to prepare and label. These cells which have a more efficient uptake of Indium and long biological survival (up to 9 days) are capable of detecting a wide range of lesions. The use of labelled granulocytes with a short biological survival (about 4 hours) is restricted to haematological investigations and diagnosis of acute bacterial infections or necrosis.
Platelet Activating Factor (PAF)-Induced Aggregation And Release Responses In Vitro In Three Species
Authors: Cargill D I , Cohen D S , Van Valen R , Lee M L , Saunders R N
Published: July 2018

PAF-induced aggregation in human, rabbit and guinea pig platelets was studied, along with concomitant dense granule (ATP) release. Platelet rich plasma (PRP) was prepared from citrated whole blood (9:1) and adjusted to 5 × 10° plt/ ml. Aggregation and release assays were performed in a Chronolog Lumi-Aggregometer. PAF was dissolved in Tris- Tyrode-Albumin (0.25%) buffer, pH 7.4. ATP standards were run prior to each assay.

ATP release was most sensitive in the guinea pig (range 10-10 (max) to 10-11(min) M PAF), less in the rabbit (10-8 to 10-9M PAF) and least sensitive to PAF in human platelets. In human platelets PAF elicited a biphasic release of ATP, the first immediately following addition of PAF and the second at_4-5 min. Release was induced in human platelets by 2 ß l0-7 to 6 × 10-7M PAF.

Throughout the range of PAF concentrations which caused a dose-related release of ATP in guinea pig platelets, platelet aggregatory response remained constant (85% or more). Similar findings were observed in rabbit platelets.

Human PRP exhibited biphasic release and irreversible aggregation at 10-6M PAF. At 10-7M the initial phase of ATP release disappeared, while a biphasic aggregatory response remained, accompanied by a secondary release pattern. Aspirin (ASA 200 μM final concentration) inhibited both the secondary phases of release and aggregation.

PAF-induced aggregation in rabbit and guinea pig platelets appears to be independent of the release reaction. In human platelets, PAF gives a biphasic release pattern, the second phase being inhibited by ASA. The significance of the novel first phase of release is being studied.
Measurement Of 1-14C Arachidonic Acid Metabolism By Cultured Human Vascular Endothelial Cells And Platelets
Authors: Willems Ch , Laanen P J , Pool G A , de Groot Ph G , van Mourik J A , van Aken W G
Published: July 2018

To investigate the relative sensitivity of endothelial cells and platelets towards aspirin (ASA), a method was designed which allows the measurement of prostaglandin (PG) metabolism using [l-14C] arachidonic acid (AA) as precursor. When HEC were incubated with 20 μM AA for 24h at 37°C in the presence of serum, about 15-20% of the label was incorporated into phospholipids whereas 3-4% was present in the neutral lipid fraction. No PG formation could be measured, even after stimulation of the phospholipase system by thrombin or ionophore A 23187. When HEC were incubated for 20 min with AA in the absence of serum, about 8-10% of the label was incorporated into PGs (6-keto PGF1α, 2%; PGE2, 1%; PGF2α, 4%). Platelets were prelabeled with AA, in buffer and the phospholipase system could be stimulated by thrombin (measured as the formation of TXB2, HHT and HETE). Using the above methods, the IC50 of ASA (30 min, 37°C) was 4.7±2.4μM for HEC and 10.1±2.4μM for platelets (p>0.1). Preincubation with 5μM ASA resulted in 50% inhibition of PG synthesis after 30±8 min with HEC and after 40±5.6 min with platelets (p#x003E;0.1). Moreoever when HEC and platelets were combined, incubated with ASA and subsequently tested separately no difference in dose-response curves could be demonstrated. These results indicate that the sensitivity of HEC and platelets to ASA is similar.
Pathways Of Phospholipid Bound Arachidonic Acid To Prostaglandin-Like Metabolites In Rat Platelets After Aggregation
Authors: Zijlstra F J , van Vliet H H D M , Vincent J E
Published: July 2018

The distribution of [l-14c] arachidonic acid (AA) over phospholipids (PL) and neutral lipids and the formation of labelled thromboxane B2 (TxB2), 12L-hydroxy-5,8,10 hepta- decatrienoic acid (HHTJand 12L-hydroxy-5,8,10,14 eicosa- tetraenoic acid (HETE) was measured in rat platelets at different times after the induction of aggregation by collagen. PL, neutral lipids and prostag1andin-(PG) like substances were separated by several systems of TLC and the radioactivity determined in the individual compounds.

Platelet PL were labelled with [l-14c] AA by 10 min incubation and the platelets washed to remove free AA.

After aggregation, there is rapid decrease in the amount of labelled phosphatidy1-inositol (PI) in the first 5 min and a slower decrease in that of phosphatidylcholine (PC) lasting about 20 min. There are no consistent changes in the amount of labelled phosphatidyl-ethanol amine. In the neutral lipids, only a decrease in the amount of labelled diglycerides occurs during the first 5 min. The amounts of TxB2 and HHT formed augment, rapidly after the aggregation and reach a maximum value after 5 min. The increase in HETE is much slower with a maximum after 20 min.

These data indicate that the AA, used in the formation of TxB2 and HHT is probably derived from PI after degradation by a phospholipase C-like enzyme to diglycerides.

On the contrary, the time course suggests that the AA transformed via the lipoxygenase pathway to HETE is originally present in PC and released by the action of phospholipase A2. This would explain the differences in the formation.
Strontium Ions Stimulate The Metabolism Of Phosphoinositides In Human Blood Platelets
Authors: Best L C , Bone E A , Russell R G G
Published: July 2018

We have previously shown that strontium ions (Sr2+ ) cause platelet 5-hydroxytryptamine secretion and thromboxane (TxB2) production in parallel. Here, we have further investigated the mechanism by which Sr2+ causes platelet TXB2 production and platelet activation by examining phospholipid metallism in washed human platelets in response to Sr2+ . Platelet phospholipid pools were labelled by preincubation with 3H- glycerol. Phospholipids and neutral lipids were extracted with chloroform-methanol and resolved bytwo-dimensional thin layer chromatography. Sr2+ (l-8mM) produced a marked depletion in platelet phosphatidyl inositol (PI) with a maximal effect at 10 minutes. The major products corresponded to polyphosphoinositides (PPIs) and lysophosphatidyl inositol (LPI). No significant changes in other phospholipids were detected. Similarly, no consistent effects were observed on mono or diglyceride levels. PI breakdown in response to Sr2+ was inhibited by Ca , prostaglandin E1, and trifluoperazine, suggesting the possible involvement of calmodulin in Sr2+ action. PI break-down was also partially inhibited by aspirin and indomethacin but not by imidazole. We suggest that Sr2+ induces platelet secretion and TXB2 production by causing the specific break down of PI, presumably by activating a phospholipase. The significance of the observed effect upon PPI metabolism is unknown, although PPI turnover may be closely associated with the control of calcium fluxes. Thus, Sr2+ may mimick a rise in cytosol Ca2+ in the platelet which is thought to accompany platelet activation.
Anticoagulant And Antithrombotic Properties Of D-PHE-PRO-ARG-H /1/, BOC-D-PHE-PRO-ARG-H /2/, BZ-D-ALE-PRO-ARG-H /3/ And β-D-Phenyl-Lactyl-PRO-ARG-H /4/
Authors: Bagdy D , Barabás É , Banusz S , Széll E , Boaor I
Published: July 2018

A series of tripeptide derivatives was synthetized
and studied for blood clotting inhibition in vitro and in vivo experiments. Whole blood clotting time /WBCT/, activated partial thromboplastin time /APTT/, thrombin time /TT/ and prothrombin time /PT/ were measured in Thrombelastograph and Coagulometer, resp. using heparin and hirudin as reference substances. The inhibition by /1-4/ of platelet aggregation /PA/ was tested in Chrono-Log Aggregometer. The anticoagulant action of /1-4/ in vivo was investigated in mice, rats, rabbits, dogs and monkeys, resp. by different ways of administration. /1/ was found to be a non competitive inhibitor of thrombin according to Dixon plots /Ki = 7.5 × l0-8 M /. /1-4/ inhibited the thrombin induced PA specifically. By giving to animals i.v.,i.m.,s.c. and orally, resp. /1-4/ proved to be effective in all species and the anticoagulant action was dose dependent. In the course of continuous i.v.infusions to conscious rabbits and narcotized dogs, resp. /3-4 mg pro kg/hr for 3 to 6 hrs / WBCT, APTT and TT prolonged to 3-5 times of the starting values. The anticoagulant effect appeared 15 min after starting and returned to normal within 1-2 hrs after stop the infusions. No change either in blood pressure or in respiration could be observed during the infusions. ED50 and LD50 of i.v. infusion in conscious rabbits was found to be 1.3 and 58 mg, resp.. Our results suggest that /1-4/ represent powerful anticoagulant and antithrombotic effects and can be regarded as a new tyoe of anticoagulants.
Experience Of Control Of The Initiation Of Coumarol Therapy With An Amidolytic Assay Of Factor X
Authors: Egberg N , Johnsson H
Published: July 2018

Most authors agree that probably any of the vitamin K dependant factors can be used for control of coumarol therapy at steady state of treatment. However, during initiation of coumarol therapy poor correlations have been found between one stage prothrombin assays and specific factor analyses (II and X) and thus the usefulness of the specific assays in the initial phase of treatment has been questioned.

Purpose of study To investigate if a specific factor X analyses could be used for control of the initiation of coumarol (warfarin) therapy.

Methods Thrombotest (Nyegaard AS, Oslo). Amidolytic factor X assay according to Bergström and Egberg.

Patients admitted to the hospital because of venous thrombosis and/or pulmonary embolism. At present 13 patients (9 men, 4 women), age 36-81 years.

Design of study Heparin therapy was started as soon as the diagnosis was assigned. Warfarin treatment was commenced on the following day. Thrombotest and factor X was determined before and daily for 6 days after coumarol therapy was started. Only the results from the factor X analyses were given to the doctor in charge of the patients for monitoring of the coumarol therapy.

Results Thrombotest and factor X values generally fell in parallell except when high concentrations of heparin in the blood samples inhibited the Thrombotest method. The correlation between Thrombotest and factor X in 10 patients was good (r 0.81-0.99, mean 0.94).(Three patients were excluded for different reasons). The doctor in the ward found it as easy to monitor coumarol treatment with the factor X assay as with the Thrombotest. No bleeding or thromboembolic complications occurred.

Conclusion The amidolytic factor X assay could be used for monitoring coumarol treatment in the initial phase of treatment. The absence of sensitivity to heparin was regarded an advantage to the Thrombotest method.
Reactivity Of Human Fibrinogen Adsorbed On Two Different types Of Hemodialysis Membranes
Authors: Chuang H Y K , Sharpton T R , Mohammad S F , Sharma N C , Mason R G
Published: July 2018

Cuprophane and polyacrylonitrile are two common materials used as hemodialysis membranes. Examination by both phase contrast and electron microscopy of both types of material after clinical ex vivo use with uremic patients indicated that cellular deposition was extensive on Cuprophane but not on polyacrylonitrile. In vitro studies with 125I-labeled human fibrinogen or immunpglobulin G (IgG) showed that the adsorption of fibrinogen and IgG was greater on polyacrylonitrile than on Cuprophane. These results appear to be in conflict with the general concept that materials that adsorb large quantities of protein, especially fibrinogen and IgG, will promote greater adhesion of platelets and leukocytes. Further studies of fibrinogen adsorbed on polyacrylonitrile surfaces indicated that the adsorbed fibrinogen: (a) was not readily accessible for reaction with 125I-labeled antifibrinogen IgG, (b) was not desorbed readily from the surface, (c) was not appreciably displaced by other plasma proteins such as albumin, IgG, except fibrinogen, and (d) did not promote the adhesion of 51Cr-labeled platelets or polymorphonuclear leukocytes. These data indicate that fibrinogen, though present in high concentration on polyacrylonitrile, may adsorb in a biologically inactive form. This could be due to either the direct involvement of fibrinogen active sites in the interaction of the protein molecule with polyacrylonitrile or a conformational change in the fibrinogen molecule upon adsorption. Our observations suggest that the thrombogenicity of an artificial surface may not be assessed by the type and amount of various protein adsorbed but is likely determined by the reactivity of specific adsorbed protein species.
Diagnostic value Of Ethanol Gelation Test (EGT) And Fibrin- (OGEN) Degradation Products (FDP) In Suspected Deep Vein Thrombosis (DVT), Correlated To The 125-1-Fibrinogen Uptake Test (FUT)
Authors: Hamborg T , Skjennald A , Godal H C
Published: July 2018

The study was performed in order to investigate the correlation between EGT, respectively FDP, and a positive FUT (FUT+), confirmed by venography. Control groups: patients 1) with a negative FUT (FOT−) in spite of DVT, 2) without DVT, because a negative venography also implies FUT−. Accordingly, FUT was not accomplished in this group.

The material consisted of 41 patients with clinically suspected DVT; 20 with and 21 without DVT. Of the patients with DVT, 9 were definitely FUT+ and 11 definitely FUT−.

EGT was applied for detection of soluble fibrin, Thrombo- Wellcotest for FDP and the Chi-squared test for statistical analysis. Blood specimens were analysed 0-1 days before isotope injection in patients with DVT, or 0-2 days before a negative venography.

Conclusions: In this material, 1) Concurrently abnormal EGT and FDP was specific for the FUT+ patients. 2) On the other hand, a positive FUT could be excluded when both parameters were normal. 3) In patients with DVT, a positive EGT was found only in the FUT+ patients. (P values in the table refers to comparison with the FUT+ group).
Automated Coagulation Factor Assays Using A Thrombin Specific Chromogenic Substrate
Authors: Duncan A , Bowie E J W , Owen C A
Published: July 2018

An automated method for specific factor assays has been developed using a novel thrombin-specific chromogenic substrate (developed by A. Aiyappa, Ph. D., Abbott Labs). For factors VIII, IX, XI and XII, the test system includes an ellagic acid activator and brain phospholipid in a calcium containing tris buffer pH 7.4. These are mixed with a plasma sample consisting of a mixture of specific factor deficient substrate and test plasma for 5 minutes and then the chromogenic substrate is added. After another 5 minutes the increase in optical density ΔOD is recorded. Normal plasma serially diluted in specific factor deficient substrate is used to construct a standard graph and test plasma results are interpolated from this.

Fifty-four comparative assays have been done for factors VIII and IX and correlated with the results obtained from conventional clotting assays. For factor VIII the correlation coefficient (r)=0.935 (N=35) and for factor IX r=0.941 (N=19). While insufficient data exists to date for factors XI and XII, these assays appear to be comparable to the VIII and IX results.

The assays for factors V, VII and X use a simplified system in that the chromogenic substrate and phospholipid are added together. The ΔOD is again recorded after 5 minutes. Test plasma results are interpolated from a standard graph constructed using serially diluted normal plasma as before. To date only the factor V assay has enough data to do a correlation with the clotting assays and r=0.953 (N=13) which is similar to the r value for the VIII and IX assays.

Because of the widespread availability of the automated analyzer used in the assay and the standardization of end point determination it provides, specific factor assays might be made more widely available outside specialized coagulation centers.